Chronic Pain Induced by Social Defeat Stress in Juvenile Mice Depends on TLR4

Abstract

A significant portion of adolescents suffer from mental illnesses and persistent pain due to repeated stress. The components of the nervous system that link stress and pain in early life remain unclear. Prior studies in adult mice implicated the innate immune system, specifically Toll-like receptors (TLRs), as critical for inducing long-term anxiety and pain-like behaviors in social defeat stress (SDS) models. In this work, we investigated the pain and anxiety behavioral phenotypes of wild-type and TLR4-deficient juvenile mice subjected to repeated SDS and evaluated the engagement of TLR4 by measuring dimerization in the spinal cord, dorsal root ganglia, and prefrontal cortex. Male juvenile (4-week-old) mice (C57BL/6J or Tlr4^-/-) underwent six social defeat sessions with adult aggressor (CD1) mice. In WT mice, SDS promotes chronic mechanical allodynia and thermal hyperalgesia assessed via von Frey testing and the Hargreaves test, respectively. In parallel, the stressed WT mice exhibited transient anxiety-like behavior and long-lasting locomotor activity reduction in the open-field test. Tlr4^-/--stressed animals were resistant to the induction of pain-like behavior but had a remnant of anxious behavior, spending less time in the center of the arena. In WT SDS, there were concordant robust increases in TLR4 dimerization in dorsal root ganglia macrophages and spinal cord microglia, indicating TLR4 activation. These results suggest that the chronic pain phenotype and locomotor impairment induced by SDS in juvenile mice depends on TLR4 engagement evidenced by dimerization in immune cells of the dorsal root ganglia and spinal cord.

Keywords: TLR4; dorsal root ganglia; pain; social stress; spinal cord.

Figure 1

SDS promotes pain phenotype and reduction in locomotor activity. (A) Experimental timeline, SDS protocol, and behavior evaluation time points. (B) Paw mechanical threshold by von Frey filament of SDS and no-SDS groups, days 0, 7, 11, and 21. (C) Paw thermal threshold by Hargreaves test of SDS and no-SDS groups, days 0, 7, 11, and 21. (D) Open-field test: total distance traveled in the arena (cm). (E) Mean body velocity of each animal (cm/s). (F) Maximum body acceleration (cm/s²). (G) Mean body mobility (%). Data represent the mean ± SEM. Two-way ANOVA followed by Bonferroni’s multiple comparisons test for (A,B). One-way ANOVA followed by Bonferroni’s multiple comparisons test for (D–G). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns means non-significant. n = 6 (SDS); n = 7 (no-SDS).

Figure 2

SDS reduces the spontaneous interest in exploring the arena on day 10. (A–D) Representative tracking heatmap image of four mice in the open-field arena. Color spectrum gradient: blue, low intensity; red, high intensity. (A) The no-SDS group and (B) the SDS group, on day 10. (C) The no-SDS group and (D) the SDS group, on day 17. (E) Frequency to the center of the arena. (F) Time spent in the center. (G) Frequency of visits to the corner of the arena. (H) Time spent in the corners. Ligh-and-dark test: (I) cumulative time spent in the bright chamber; (J) time until the first entrance of the bright chamber; (K) frequency of entries to the bright chamber. Data represent the mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparisons test: * p < 0.05; ** p < 0.01; n = 6 (SDS); n = 7 (no-SDS); and ns means non-significant.

Figure 3

SDS increases TLR4 dimers of spinal cord microglia and DRG macrophages. (A) TLR4 dimers (%) in SDS and no-SDS groups of microglia (CD11b⁺ population) and (B) macrophages (CD11b⁺/F4-80⁺ population) on day 22 of the protocol. (C) Gating strategy for lumbar spinal cord cells, first live cells were gated (APC-Cy7 live/dead Ghost dye); second, microglia population (PercpCy 5.5-conjugated CD11b+), lastly—TLR4 monomers (PC7-conjugated TLR4/MD2) or TLR4 total (APC-conjugated total TLR4). (D) Gating strategy for dorsal root ganglia cells, first live cells were gated (APC-Cy7 live/dead Ghost dye); second, microglia population (PercpCy 5.5-conjugated CD11b+), third macrophages population (488-conjugated F4-80) and lastly—TLR4 monomers (PC7-conjugated TLR4/MD2) or TLR4 total (APC-conjugated total TLR4). Data represent mean ± SEM, t-test, SDS vs. no-SDS, * p < 0.05, ** p < 0.01, n = 6 (SDS), and n = 6 (no-SDS).

Figure 4

SDS did not promote the pain phenotype Tlr4^-/- in mice. (A) Experimental timeline, SDS protocol, and behavior evaluation time points. (B) Paw mechanical threshold by von Frey filament of SDS and no-SDS KO mice at days 0, 7, 11, and 21. (C) Paw thermal threshold by Hargreaves test in SDS and no-SDS groups KO mice at days 0, 7, 11, and 21. Open-field test, in (D) total distance traveled in the arena (cm), in (E) time spent in the center, in (F) frequency of visit in corners of the arena, and (G) frequency of visit in the center area. (H–K) Locomotor activity parameters of SDS and no-SDS in KO mice. Data represent the mean ± SEM. Two-way ANOVA followed by Bonferroni’s multiple comparisons test for (B,C). One-way ANOVA followed by Bonferroni’s multiple comparisons test for (D–K). * p < 0.05, ** p < 0.01, n = 4 (SDS), n = 4 (no-SDS), and ns means non-significant.