Temporal and sex differences in the role of BDNF/TrkB signaling in hyperalgesic priming in mice and rats

Abstract

Brain-derived neurotrophic factor (BDNF) signaling through its cognate receptor, TrkB, is a well-known promoter of synaptic plasticity at nociceptive synapses in the dorsal horn of the spinal cord. Existing evidence suggests that BDNF/TrkB signaling in neuropathic pain is sex dependent. We tested the hypothesis that the effects of BDNF/TrkB signaling in hyperalgesic priming might also be sexually dimorphic. Using the incision postsurgical pain model in male mice, we show that BDNF sequestration with TrkB-Fc administered at the time of surgery blocks the initiation and maintenance of hyperalgesic priming. However, when BDNF signaling was blocked prior to the precipitation of hyperalgesic priming with prostaglandin E₂ (PGE₂), priming was not reversed. This result is in contrast to our findings in male mice with interleukin-6 (IL6) as the priming stimulus where TrkB-Fc was effective in reversing the maintenance of hyperalgesic priming. Furthermore, in IL6-induced hyperalgesic priming, the BDNF sequestering agent, TrkB-fc, was effective in reversing the maintenance of hyperalgesic priming in male mice; however, when this experiment was conducted in female mice, we did not observe any effect of TrkB-fc. This markedly sexual dimorphic effect in mice is consistent with recent studies showing a similar effect in neuropathic pain models. We tested whether the sexual dimorphic role for BDNF was consistent across species. Importantly, we find that this sexual dimorphism does not occur in rats where TrkB-fc reverses hyperalgesic priming fully in both sexes. Finally, to determine the source of BDNF in hyperalgesic priming in mice, we used transgenic mice (Cx3cr1^CreER × Bdnf^flx/flx mice) with BDNF eliminated from microglia. From these experiments we conclude that BDNF from microglia does not contribute to hyperalgesic priming and that the key source of BDNF for hyperalgesic priming is likely nociceptors in the dorsal root ganglion. These experiments demonstrate the importance of testing mechanistic hypotheses in both sexes in multiple species to gain insight into complex biology underlying chronic pain.

Keywords: BDNF; Hyperalgesic priming; IL6; Sex differences; TrkB.

Fig. 1

TrkB-fc blocks mechanical hypersensitivity and hyperalgesic priming induced by plantar incision. Male mice that received an intrathecal (i.t.) injection of TrkB-fc (30 ng) on the day of plantar incision surgery exhibited reduced mechanical hypersensitivity (A; two-way ANOVA; F (1, 90) = 58.57, P < 0.0001; Bonferroni’s ^**p < 0.01; Dunnett’s from BL-Veh-D1-10 p < 0.0001; TrkB-Fc D1-6 p < 0.0001) but not PGE₂ (100 ng)-induced hyperalgesic priming (B; two-way ANOVA; F (1, 30) = 2.753, P = 0.1075) compared to vehicle. Male mice that received two i.t. injections of TrkB-fc (30 ng) on the day of plantar incision surgery and 24 hr after also had decreased mechanical hypersensitivity (C; two-way ANOVA; F (1,90) = 95.09, P < 0.0001; Bonferroni’s ^**p < 0.01; Dunnett’s from BL-Veh-D1-13 p < 0.01; TrkB-Fc D1-2 p < 0.05) and PGE₂ (100 ng)-induced hyperalgesic priming (D; two-way ANOVA; F (1, 27) = 15.27, P = 0.0006; Bonferroni’s ^*p < 0.05) compared to vehicle.

Fig. 2

ANA-12 blocks mechanical hypersensitivity and hyperalgesic priming induced by plantar incision. Male mice that received intraperitoneal (i.p.) injections of ANA-12 (1 mg/kg), a TrkB antagonist, the day of plantar incision surgery, 24, and 48 h after showed reduced mechanical hypersensitivity (A; two-way ANOVA; F (1, 81) < 0.0001; Bonferroni’s ^*p < 0.05.) and hyperalgesic priming (B; two-way ANOVA; F (1, 27) = 38.62, P < 0.0001; Bonferroni’s ^**p < 0.01) compared to vehicle.

Fig. 3

TrkB-fc, but not ANA-12, reverses plantar incision-induced hyperalgesic priming. Male mice underwent plantar incision surgery and received 1 or 2 i.t. injection (s) of TrkB-fc (30 ng) (A) or 2 i.p. injections of ANA-12 (1 mg/kg) (B) after recovering. Mice that received TrkB-fc reversed the priming effect induced by plantar incision (A; two-way ANOVA; F (2, 42) = 2.164, P = 0.1276; Bonferroni’s ^*p = 0.0284, ^**p = 0.0082). However, ANA-12 did not reverse PGE₂ (100 ng)-precipitated hyperalgesic priming (B; two-way ANOVA; F (1, 30) = 2.696, P = 0.1111).

Fig. 4

BDNF/TrkB signaling plays a sexually-dimorphic role in IL6-induced hyperalgesic priming in mice. A) Both male and female mice received an intraplantar (i.pl.) injection of IL6 (0.1 ng) and were tested for mechanical hypersensitivity at 3, 24, 48, and 72 h. IL6 induced equal levels of mechanical hypersensitivity compared to BL in both male and female mice (A; two-way ANOVA; from BL F (4, 95) = 43.4, P < 0.0001; between sexes F (1, 95) = 4.722, P = 0.0323; Bonferroni’s ^##p = 0.001). After recovering, female mice received a single i.t. injection of vehicle or TrkB-fc (30 ng) along with male mice. TrkB-fc significantly reversed IL6-induced hyperalgesic priming in male mice, but not in female mice (B; two-way ANOVA; vs female vehicle, F (2, 54) = 26.82, P < 0.0001; vs female TrkB-fc, F (1, 33) = 33.63, P < 0.0001; ^****p < 0.0001).

Fig. 5

BDNF/TrkB signaling plays a role in IL6-induced hyperalgesic priming in both male and female rats. Male and female rats received an i.p. injection of IL6 and were observed for mechanical hypersensitivity. A) While IL-6 (0.1 ng) induced equal amounts of mechanical hypersensitivity in both male and female rats compared to BL at 3 hr post injection, male rats recovered faster compared to females (two-way ANOVA; from BL, F (6, 154) = 81.53, P < 0.0001; ^####p < 0.0001; male vs female rats, F (1, 154) = 8.633, P < 0.0038; ^**p < 0.01). B) After rats recovered, male and female rats received either a i.t. injection of TrkB-fc (30 ng) or Vehicle. Subsequently, PGE₂ was used to precipitate hyperalgesic priming. TrkB-fc was able to reverse IL6-induced priming in both male and female rats (three-way ANOVA; Time × Sex × Treatment F (2, 60) = 6.429, P = 0.003; Bonferroni’s ^****p < 0.0001).

Fig. 6

Male and female mice with BDNF eliminated from microglia show no differences in IL6-induced mechanical hypersensitivity and hyperalgesic priming. A) Tamoxifen-injected male and female Cx3cr1^Cre × BDNF^−/− and Cx3cr1^Cre × BDNF^+/+ mice received an i.pl. injection of IL6 and showed no differences in mechanical hypersensitivity (two-way ANOVA; F(3, 115) = 1.339, P = 0.2653) or PGE₂ (100 ng) hyperalgesic priming (B; two-way ANOVA; F (3, 66) = 1.209, P = 0.3135).

Supplementary figure 1