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. 2024 Jul 30;17(847):eadn8936.
doi: 10.1126/scisignal.adn8936. Epub 2024 Jul 30.

Peripheral macrophages contribute to nociceptor priming in mice with chronic intermittent hypoxia

Samuel B Chivers  1 , Mary Ann Andrade  2 , Robert J Hammack  2 , John Shannonhouse  1 , Ruben Gomez  1 , Yan Zhang  1 , Brian Nguyen  1 , Pankil Shah  3 , Yu Shin Kim  1 , Glenn M Toney  2 , Nathaniel A Jeske  1   2   4
Affiliations

Peripheral macrophages contribute to nociceptor priming in mice with chronic intermittent hypoxia

Samuel B Chivers et al. Sci Signal. .

Abstract

Obstructive sleep apnea (OSA) is a prevalent sleep disorder that is associated with increased incidence of chronic musculoskeletal pain. We investigated the mechanism of this association in a mouse model of chronic intermittent hypoxia (CIH) that mimics the repetitive hypoxemias of OSA. After 14 days of CIH, both male and female mice exhibited behaviors indicative of persistent pain, with biochemical markers in the spinal cord dorsal horn and sensory neurons of the dorsal root ganglia consistent with hyperalgesic priming. CIH, but not sleep fragmentation alone, induced an increase in macrophage recruitment to peripheral sensory tissues (sciatic nerve and dorsal root ganglia), an increase in inflammatory cytokines in the circulation, and nociceptor sensitization. Peripheral macrophage ablation blocked CIH-induced hyperalgesic priming. The findings suggest that correcting the hypoxia or targeting macrophage signaling might suppress persistent pain in patients with OSA.

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Figures

Figure 1:
Figure 1:. CIH-induced acute and hyperalgesic priming behavior.
(A) Changes in mechanical sensitivity thresholds in male and female mice after 7 or 14 days of CIH or normoxia. M, male; F, female. Data are means ± SEM from n=4 mice per sex per condition, presented as percent change from baseline day 0. No statistical significance between any group, by two-tailed student’s t-test. (B) Data in (A), analyzed with the sexes combined in each treatment group. n=8 mice per group; NS, no statistical significance, tested as in (A). (C) Treatment and behavior paradigm. (D) Male and female mice conditioned for 14 days under CIH or normoxia, followed by intraplantar injection of PGE2 (100 ng, red triangle) were then tested 15 min to 96 hours afterward in a blinded fashion for their mechanical sensitivity threshold as in (A). Data are means ± SEM from n=8 mice per group, of mixed sex (4 each), presented as % of baseline. Φp<0.0001 by two-way ANOVA with Tukey’s post-hoc test for pairwise comparison, for both comparisons of male CIH vs normoxic groups, and female CIH vs normoxic groups.
Figure 2:
Figure 2:. Effects of CIH on biochemical markers of hyperalgesic priming.
(A to F) Dorsal root ganglia (DRG) and spinal dorsal horn (DH) tissues from mice isolated after the 14-day CIH or normoxic (Norm) paradigms were analyzed by Western blotting for markers of hyperalgesic priming. Representative blots (A, C and E) and analysis (shown as means ± SEM; B, D, and E) were each from n= 4 male mice per group. NS = no significance, *p<0.05, and **p<0.01 by unpaired t-test (p=0.0038 for p-PKCε, p=0.0044 for p-p65 NF-κB, p=0.0100 for p-p38).
Figure 3:
Figure 3:. Sleep fragmentation does not Induce hyperalgesic priming.
(A) Male mice (n=6-8/group) tested for mechanical threshold) following 7 or 14 days of sleep fragmentation (SF), alone or followed by intraplantar injection of PGE2 (100ng, red triangle, left graph). Mechanical threshold to von Frey filaments, using the up-down method, at 15 min and 1 to 96 hours after injection were observed in a blinded manner. Data are mean ± SEM from 6 to 8 mice per group, presented as % of baseline. Two-way ANOVA indicated no statistically significant difference between groups, p=0.6492. (B) Representative tracking of movement during elevated plus maze testing of male mice (n=4 to 8 mice per group) following control treatment or a 7-day or 14-day sleep fragmentation protocol. (C) The amount of time mice from (B) spent in closed arms, open arms, and center zone of the maze, assessed in a manner that was blinded to the conditions. Data are means ± SEM. Closed arms: NS, not significant by Kruskal-Wallis test (p=0.2177, normality assumption rejected). Open arms: *p<0.05 by one-way ANOVA [F*888 (2,15)=6.767, p=0.0080]. Center zone: *p<0.05 by one-way ANOVA [F (2,15)=4.864, p=0.0235].
Figure 4:
Figure 4:. CIH Increases responsivity of DRG neurons.
(A) Real-time responsivity to 25mM KCl stimulus in DRG neurons cultured from male mice that had undergone 14 days of CIH (green) or normoxia (grey). Shown is a representative trace from cultured DRG neurons in each condition, assessed 2 hours after isolation into culture. (B) Cumulative summary of peak responses from DRG neurons described in (A), displayed as mean from n=191 neurons isolated from CIH mice and 240 neurons isolated from normoxic mice. *p<0.05 by unpaired t-test (p=0.0113). (C) Estimation plot of data in (B) to demonstrate magnitude of effect. (D to L) In vivo Ca2+ imaging in GCaMP reporter mice exposed to chronic intermittent hypoxia (CIH) or normoxia for 14 days followed by intraplantar injection of PGE2, and stimulated 2 hours later with a 100g press on hind paw (D to F) or a 50°C (G to I) or 10°C (J to L) challenge to the plantar side of the hind paw. Ca2+ transients overlayed with averaged Ca2+ transient curve illustrated in the left panels; total number of responding neurons in the right, where mean ± SEM are shown from n=6 mice per condition, 3 per sex. Males are represented by black triangles, females by colored triangles. NS=no significance, **p<0.01, and ***p<0.001 by unpaired t-test (p=0.0009 for 100g press, p=0.0069 for 50°C heat, p=0.0677 for 10°C heat).
Figure 5:
Figure 5:. Effect of CIH on circulating cytokines.
(A to F) Retro-orbital venous blood was drawn from mice before [day (d) 0] and 7 and 14 days after CIH or normoxia exposure. Blood serum was quantified for IL-4 (A), IL-6 (B), GM-CSF (C), IL-10 (D), IL-2 (E), and IL-1β (F) in a manner that was blinded to the conditions. Data are mean ± SEM from n = 6 mice, 3 per sex. Males are represented by black triangles, females by colored triangles. *p<0.05, **p<0.01, and ****p<0.0001 by two-way ANOVA, Tukey’s post-hoc for pairwise comparison (IL-6: exact p-value <0.0001 not given in Prism; GM-CSF: p=0.0065 normoxic day 14 vs. CIH day 14, p=0.0341 CIH days 7 vs. 14; IL-1β: p=0.0285 normoxic day 14 vs. CIH day 14, p=0.0201 normoxic day 7 vs. CIH day 7). (G to I) Concentration of GM-CSF, IL-1β, IL-2, and IL-4 in DRG (G) sciatic nerve (H), and spinal cord (I) tissue samples extracted from mice after 14 days of normoxia or CIH exposure, quantified in a condition-blinded manner. For DRG and sciatic nerve tissues, n=4 samples, each pooled from 2 mice of the same sex, due to low-level cytokine detection. For spinal cord tissues, n=8 samples, 1 from each of 8 mice, 4 per sex. Males are represented by black circles, females by colored circles. Data are mean ± SEM. **p<0.01 and ***p<0.0001 by two-way ANOVA, with Sidak’s post hoc multiple comparison analyses (DRG GM-CSF p=0.0030, sciatic nerve GM-CSF p<0.0001).
Figure 6:
Figure 6:. FACS of macrophage cells in CIH peripheral tissues.
(A) FACS analysis of infiltrating macrophages in samples of skin, sciatic nerve, DRG, and circulating blood from mice 14 days after normoxia or CIH, determined by PE-F4/80 staining (green counts, A to H) assessed in a blinded manner. Plots are representative, and quantified data (B) are means ± SEM from n = 6 male mice per condition. NS=no significance, *p≤0.05 by unpaired t-test. (Sciatic, normoxic vs CIH p=0.0500; DRG, normoxic vs CIH p=0.0438).
Figure 7:
Figure 7:. DRG macrophage expression in MaFIA mice with CIH.
(A and B) L4-L6 DRG tissues from male MaFIA mice (n=4) treated with saline or AP20187 (AP) were dissected for sectioning and immunohistochemical analysis of macrophage infiltration. F4/80 (red), macrophage marker; NeuN (green), neuronal marker; DAPI (blue), nuclear marker. Images in (A) are representative; scale bar, 30μm. Data in (B) show macrophage density relative to neurons in multiple slices from each mouse, totaling 19 distinct images quantified per treatment condition (overlayed the mean ± SEM). ****p<0.0001 by Mann-Whitney U test, normality assumption rejected. (C) Change in mechanical sensitivity thresholds in male and female MaFIA mice treated with saline or AP20187 (AP) after 14 days of CIH or normoxia followed by intraplantar injection of PGE2 (red triangle). Sensitivity was assessed with the von Frey filament up-down method and recorded in a manner that was blinded to the condition. Data are mean ± SEM from n=6 mice (3 males and 3 females, data pooled) per treatment, presented as % of baseline day 0. Φp<0.0005 by unpaired t-tests (at 2 hours, p=0.000051; 8 hours, p=0.000087; 24 hours, p=0.00024; 48 hours, p=0.00057; and 96 hours, p=0.000147). (D) Baseline mechanical withdrawal thresholds, in grams (g), for WT mice (n=8, 4 from each sex) and MaFIA mice treated with saline (MaF-Sal) or AP20187 (MaF-AP) (each n=6, 3 from each sex). Males are represented by black triangles, females by colored triangles. Data are mean ± SEM. NS=no significance vs. WT by one-way ANOVA with Bonferroni correction. (E to J) Serum levels of IL-4 (E), IL-6 (F), GM-CSF (G), IL-10 (H), IL-2 (I), and IL-1β (J) in retro-orbital venous blood drawn from MaFIA mice treated with saline or AP20187 before [0 days (d)] and 7 and 14 days after CIH exposure. Data are mean ± SEM from n = 5 mice (3 males, 2 females) per treatment condition. Males are represented by black triangles, females by colored triangles. NS=no significance, **p<0.01, and ***p<0.005 by unpaired t-test (IL-6, p=0.0001 MaFIA-Sal-CIH day 0 vs 14; GM-CSF, p=0.0003 MaFIA-Sal-CIH day 0 vs 14; IL-1β, p=0.0026 MaFIA-Sal-CIH day 0 vs 14).
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