Anti-hyperalgesic and anti-inflammatory effects of 4R-tobacco cembranoid in a mouse model of inflammatory pain

Abstract

4R is a tobacco cembranoid that binds to and modulates cholinergic receptors and exhibits neuroprotective and anti-inflammatory activity. Given the established function of the cholinergic system in pain and inflammation, we propose that 4R is also analgesic. Here, we tested the hypothesis that systemic 4R treatment decreases pain-related behaviors and peripheral inflammation via modulation of the alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in a mouse model of inflammatory pain. We elicited inflammation by injecting Complete Freund's Adjuvant (CFA) into the hind paw of male and female mice. We then assessed inflammation-induced hypersensitivity to cold, heat, and tactile stimulation using the Acetone, Hargreaves, and von Frey tests, respectively, before and at different time points (2.5 h - 8d) after a single systemic 4R (or vehicle) administration. We evaluated the contribution of α7 nAChRs 4R-mediated analgesia by pre-treating mice with a selective antagonist of α7 nAChRs followed by 4R (or vehicle) administration prior to behavioral tests. We assessed CFA-induced paw edema and inflammation by measuring paw thickness and quantifying immune cell infiltration in the injected hind paw using hematoxylin and eosin staining. Lastly, we performed immunohistochemical and flow cytometric analyses of paw skin in α7 nAChR-cre::Ai9 mice to measure the expression of α7 nAChRs on immune subsets. Our experiments show that systemic administration of 4R decreases inflammation-induced peripheral hypersensitivity in male and female mice and inflammation-induced paw edema in male but not female mice. Notably, 4R-mediated analgesia and anti-inflammatory effects lasted up to 8d after a single systemic administration on day 1. Pretreatment with an α7 nAChR-selective antagonist prevented 4R-mediated analgesia and anti-inflammatory effects, demonstrating that 4R effects are via modulation of α7 nAChRs. We further show that a subset of immune cells in the hind paw expresses α7 nAChRs. However, the number of α7 nAChR-expressing immune cells is unaltered by CFA or 4R treatment, suggesting that 4R effects are independent of α7 nAChR-expressing immune cells. Together, our findings identify a novel function of the 4R tobacco cembranoid as an analgesic agent in both male and female mice that reduces peripheral inflammation in a sex-dependent manner, further supporting the pharmacological targeting of the cholinergic system for pain treatment.

Keywords: 4R; Hyperalgesia; Inflammatory pain; Macrophage; Paw edema; Persistent analgesia; Tobacco cembranoid; α7 nicotinic acetylcholine receptors.

Fig. 1

Systemic administration of 4R tobacco cembranoid reduces inflammation-induced peripheral hypersensitivity (a) Chemical structure (left panel) and 3D molecular model (right panel) of 4R-tobacco cembranoid. Oxygen, hydrogen, and carbon atoms are colored red, white, and grey, respectively. (b) Experimental timeline for acetone (cold), Hargreaves (heat) and von Frey (tactile) tests in CFA-injected and non-injected hind paws before and at different times after 4R (1, 6 and 15 mg/kg) or vehicle administration. (c) Acetone response score, (d) withdrawal latency to heat stimulation (e) withdrawal threshold to tactile stimulation. All values are expressed as mean ± SEM. n = 4–9 animals per treatment and test. Two-way ANOVA followed by posthoc Tukey’s multiple comparisons test: CFA-injected paw vs uninjected paw in pre-drug vehicle (veh) conditions, p < 0.0001 (####), 4R treated CFA-injected paw vs vehicle uninjected paw, p < 0.0001 (¶ ¶ ¶ ¶); not significant = ns. Two-way ANOVA followed by post hoc Dunnett’s multiple comparisons test: CFA-injected paws in vehicle vs 4R systemic treatment of the same time-point, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Wilcoxon matched-pairs signed rank test: CFA-injected paw vs uninjected paw in pre-drug vehicle (veh) conditions, p < 0.01 (††), Non-parametric Mann–Whitney multiple comparisons test followed by Holm-Šídák method; CFA-injected paws in vehicle vs 4R systemic treatment of the same time-point, adjusted p < 0.01 (‡‡), adjusted p < 0.001 (‡‡‡); vehicle (veh) uninjected paw (day 7) vs 4R 15 mg/kg CFA-injected paw (day 7), adjusted p > 0.05 (§§). Results of multiple comparisons between vehicle (veh) and 4R 1 mg/kg, 6 mg/kg or 15 mg/kg are shown in purple, teal, and orange, respectively

Fig. 2

Systemic 4R administration reduces inflammation-induced thermal but not tactile hypersensitivity in female mice (a) Experimental timeline for acetone (cold), Hargreaves (heat) and von Frey (tactile) tests in CFA-injected and uninjected hind paws at days 2 and 7 after 4R (15 mg/kg) or vehicle administration in females. Acetone response score (b, e), withdrawal latency to heat stimulation (c, f) and withdrawal threshold to tactile stimulation (d, g) on experimental days 2 and 7, respectively. All values are expressed as mean ± SEM. n = 6 animals per treatment and test. Two-way ANOVA followed by Šídák’s multiple comparison test; p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****) or Tukey’s multiple comparisons test; adjusted p < 0.0001 (§§§§). Mann–Whitney multiple comparisons test, Holm-Šídák method; adjusted p < 0.05 (#); adjusted p < 0.01 (##). Not significant = ns

Fig. 3

4R reduces inflammation-induced paw edema in male but not female mice (a) Experimental timeline for paw thickness measurements. In vivo thickness measurements of CFA-injected and non-injected hind paws before and 1–8 days after 4R (1, 6, and 15 mg/kg) or vehicle systemic administration of (b) male and (c) female mice. All values are expressed as mean ± SEM. n = 6–16 animals per treatment and sex. Two-way ANOVA (b) or Two-way RM ANOVA (c) followed by posthoc Tukey’s multiple comparisons test: CFA-injected paw vs non-injected paw in pre-drug vehicle (veh) conditions, p < 0.0001 (####); 4R CFA-injected paw vs non-injected vehicle conditions day 7, p < 0.0001 (§§§§), Two-way ANOVA followed by posthoc Dunnett’s multiple comparisons test: vehicle vs 4R systemic treatment in CFA-injected paws of the same time-point, p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Results of multiple comparisons between vehicle (veh) and 4R 1 mg/kg, 6 mg/kg or 15 mg/kg are shown in purple, teal and orange asterisks, respectively. (d) Experimental timeline for histology samples. e Representative H&E images of dermal (D) and subdermal (S) skin, open arrows point to polymorphonuclear immune cell infiltration. Scale Bar: 200 µm. Histological analysis of hind paw skin 8 days post local CFA and 7 days post 4R (f) glabrous skin thickness of the hind paws and (g) subdermal immune cell count. All values are expressed as mean ± SEM. n = 5–6 mice per treatment and sex. Two-way ANOVA followed by Šidák’s multiple comparisons test: Vehicle vs 4R, p < 0.05 (*), p < 0.01 (**)

Fig. 4

Pretreatment with an α7 nAChRs selective antagonist prevents 4R-induced reductions in inflammation-induced hypersensitivity and paw edema (a) Experimental timeline. CFA-injected and non-injected paw thickness measurements were taken in pre-drug condition. One day after CFA paw injection, MLA (10 mg/kg) or vehicle (veh) was systemically administered and 15 min after, animals received 4R (15 mg/kg) or veh treatment (s.c.). CFA-injected and non-injected paw thickness was measured 1–8 days after 4R (15 mg/kg), MLA (10 mg/kg) or vehicle administration. Acetone (cold) and Hargreaves (heat) tests in CFA-injected and non-injected hind paws were performed 7–8 days after systemic drug administration. (b, c) Acetone response scores (b) and paw withdrawal latencies in response to heat stimulation (c); Kruskal–Wallis test followed by posthoc Dunn’s multiple comparison test; saline + 4R vs. MLA + 4R; p < 0.01 (##); saline + DMSO vs. MLA + DMSO, p > 0.05, not significant (ns). (d) Thickness measurements of CFA-injected and non-injected hind paws; Two-way RM ANOVA followed by posthoc Tukey’s multiple comparisons test: CFA-injected paw vs uninjected paw in pre-drug veh conditions, p < 0.0001 (####). Two-way RM ANOVA followed by posthoc Tukey’s multiple comparisons test: CFA-injected paws in saline + DMSO vs saline + 4R systemic treatments and MLA + 4R vs saline + 4R at the same time-point, p < 0.05 (*), p < 0.01 (**). All values are expressed as mean ± SEM. n = 6 animals per treatment and test

Fig. 5

α7 nAChRs expression in hind paw macrophages (a) Experimental Timeline. Immunohistochemistry of α7^tdT/Iba1⁺ macrophages in CFA-injected and non-injected hind paw skin (subdermal) 8 days post-CFA (day 0) and 7 days post 4R (15 mg/kg) or veh treatment (s.c.) (day 8). (b) A representative image of α7^tdT and Iba1⁺ hind paw subdermal co-expression. Scale bar: 20 µm. c Cell counts normalized to the area of subdermal α7^tdT cells. (d) Cell counts normalized to the area of subdermal Iba1 + cells. (e) Cell counts normalized of colocalized α7^tdT and Iba1⁺. (f) Experimental Timeline. Flow cytometric analysis of hind paw subdermal macrophages (F4/80) and T-cells (CD3) of CFA-injected (ipsi) and non-injected (contra) hind paw skin 8 days post-CFA only. (g) Hind paw skin was enzymatically dissociated and stained with F4/80, CD3, and endogenous α7^tdT. After gating for dissociated hind paw skin cells based on forward and side scatter, cells were differentiated by their tdTomato⁺ signal, then subdivided into F4/80 and CD3 populations. (h) Analysis of overlapping of tdTomato⁺ cells that are either macrophages or T-cells in males and females

Fig. 6

4R effects as a function of time. (a) Diagram of 4R effects in males and females as a function of time. Effects on peripheral hyperalgesia (red line) and paw edema (pink line for males and purple line for females) are shown relative to previously published pharmacokinetics [68] (blue line)