Hyperalgesia in the Psychological Stress-Induced Fibromyalgia Model Shows Sexual Dimorphism Mediated by LPA1 and LPA3

Abstract

Since the initial report indicating that LPA₁ signaling plays a key role in initiating nerve injury-induced neuropathic pain (NeuP), subsequent studies using knockout mice and LPA_1/3 antagonists have demonstrated that LPA₁ and LPA₃ signaling impact NeuP and fibromyalgia (FM) models. In the present study, we identified hyperalgesia sexual dimorphism involving LPA_1/3 signaling in the intermittent psychological stress induced-related FM-like model called intermittent psychological stress (IPS)-induced generalized pain (IPGP) model where the hyperalgesia in IPGP mice was abolished in LPA₁- and LPA₃-knock-out mice. Pharmacological intervention by intraperitoneal (i.p.) treatments with the LPA_1/3 antagonist Ki16425 consistently prevented hyperalgesia. However, intracerebroventricular treatments with Ki16425 abolished hyperalgesia in male, but not female, mice. Notably, intrathecal treatments of Ki16425 did not prevent hyperalgesia. Further studies revealed that splenocytes derived from female IPGP mice could initiate hyperalgesia via adoptive transfer in naïve mice, and this effect was abolished when donor mice were pre-treated with Ki16425 (i.p.). Thus, these studies identify male-specific LPA_1/3-mediated mechanisms in the brain underlying IPGP, as well as distinct LPA-LPA_1/3-mediated peripheral immune mechanisms.

Keywords: Ki16425; LPA1; LPA3; Neurometer; clodronate liposome; empathy; fibromyalgia; knock-out mouse; psychological stress; splenocytes.

Figure 1

Reversal of male IPGP and ICGP by genetic deletion of LPA₃ or repeated i.p. treatments with LPA_1/3 antagonist. (A) Illustrative diagram of intermittent psychological stress (IPS)-induced generalized pain (IPGP) model. (B) Reversal of IPS-induced mechanical hyperalgesia by genetic deficiency of LPA₃ in male mice. Mechanical hyperalgesia at P1–P22 was observed in the paw withdrawal test. (C) Illustrative diagram of intermittent cold stress (ICS)-induced generalized pain (ICGP) model. (D) Reversal of ICS-induced mechanical hyperalgesia (P1–P19) by genetic deficiency of LPA₃ in male mice. (E) Schedule of IPS-treatments and systemic (i.p.)-treatments with Ki16425. (F–H) Time-dependent change in threshold in the IPGP model treated with vehicle or Ki16425 in the paw pressure test (F), EPW 2000 Hz (G), and EPW 250 Hz (H). (B) ** p < 0.01, two-way ANOVA followed by Tukey’s multiple comparisons test (WT-Cont, n = 6; WT-IPS, n = 6; LPA₃-KO-Cont, n = 4; LPA₃-KO-IPS, n = 5). (D) ** p < 0.01, two-way ANOVA, followed by Tukey’s multiple comparisons test (WT-Cont, n = 3; WT-ICS, n = 3; LPA₃-KO-Cont, n = 7; LPA₃-KO-ICS, n = 7). (F–H) ** p < 0.01, **** p < 0.0001, two-way ANOVA followed by Bonferroni’s multiple comparisons test (Veh, n = 3–4; Ki16425, n = 4).

Figure 2

Reversal of female IPGP by genetic deletion of LPA_1/3 or repeated i.p. treatments with Ki16425. (A) Schedule of IPS-treatments and systemic (i.p.)-treatments with Ki16425. (B–D) Time-dependent reversal of IPS-induced mechanical hyperalgesia (B) and hypersensitivity in EPW tests with 2000 Hz (C) or 250 Hz (D) by genetic deficiency of LPA₁ or LPA₃, or by Ki16425 (i.p.) in female mice. (B–D) * p < 0.05, ** p < 0.01, **** p < 0.0001, Veh vs. Ki16425 (0–19), and two-way ANOVA followed by Bonferroni’s multiple comparisons test (Veh, n = 4–6; Ki16425, n = 4–6). ^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.001, ^$$$$ p < 0.0001, n.s.; not significant, Veh vs. LPA₁-KO or LPA₃-KO, nd two-way ANOVA followed by Tukey’s multiple comparisons test (0–5; Veh, n = 4–6; LPA₁-KO, n = 4; LPA₃-KO, n = 4).

Figure 3

Male-specific reversal of hyperalgesia and hypersensitivity by repeated i.c.v. treatments with Ki16425. (A) Schedule of IPS-treatments and repeated i.c.v. treatments with Ki16425. (B–D) Reversal of IPS-induced mechanical hyperalgesia (B) and hypersensitivity in EPW tests with 2000 Hz (C) or 250 Hz (D) by Ki16425 (i.c.v.)-treatments in male mice. (E–G) Lack of reversal of IPS-induced mechanical hyperalgesia (E) and hypersensitivity in EPW tests with 2000 Hz (F) or 250 Hz (G) by Ki16425 (i.c.v.)-treatments in female mice. (B–G) ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s.; not significant, and two-way ANOVA followed by Bonferroni’s multiple comparisons test (male Veh, n = 4; female Veh, n = 5; Ki16425, n = 4).

Figure 4

Lack of reversal of hyperalgesia and hypersensitivity by i.t. treatments with Ki16425. (A) Schedule of IPS-treatments and repeated i.t. treatments with Ki16425. (B–G) Lack of reversal of IPS-induced mechanical hyperalgesia (B,E) and hypersensitivity in EPW tests with 2000 Hz (C,F) or 250 Hz (D,G) by Ki16425 (i.t.)-treatments in male (B–D) and female mice (E–G). (B–G) n.s.; not significant and two-way ANOVA followed by Bonferroni’s multiple comparisons test (male Veh, n = 4; female Veh, n = 4–8; Ki16425, n = 4–8).

Figure 5

Lack of reversal of IPS-induced hyperalgesia and hypersensitivity by clodronate liposome in the IPGP model. (A) Illustrative diagram of partial sciatic nerve injury (pSNL) model. (B) Schedule of clodronate treatments and pain tests in the partial sciatic nerve injury (pSNL) model in male mice. (C–E) Reversal of pSNL-induced mechanical hyperalgesia (C) or hypersensitivity in the EPW 2000 Hz (D) or 250 Hz (E) tests by clodronate liposome treatments. (F) Illustrative diagram of IPGP model. (G) Schedule of clodronate treatments and pain tests in the IPGP model in female mice. (H–J) Lack of reversal of the IPS-induced hyperalgesia (H) or hypersensitivity (I,J) by clodronate liposome treatments. (C–E) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and two-way ANOVA followed by Tukey’s multiple comparisons test (contra-Veh, n = 4; ipsi-Veh, n = 4; contra-Clodronate, n = 3–4; ipsi-Clodronate, n = 3–4). (H–J) n.s.; not significant and two-way ANOVA followed by Bonferroni’s multiple comparisons test (Veh, n = 4–6; Clodronate, n = 4–6).

Figure 6

Abnormal pain by splenocytes derived from IPS-treated mice. (A) Schedule of IPS treatments and repeated i.p. treatments with Ki16425 in female donor mice. (B) Experimental procedure for splenocytes injection. (C–E) Ki16425-reversible hyperalgesia (B) and hypersensitivity (C,D) in naïve female mice by adoptive transfer of splenocytes from IPS-treated donor female mice. Ki16425 (i.p.) was treated in donor mice, as described in the legend of Figure 3. (C–D) **** p < 0.0001 and two-way ANOVA followed by Tukey’s multiple comparisons test (n = 4).