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. 2022 Mar 22:16:818692.
doi: 10.3389/fnint.2022.818692. eCollection 2022.

Immunoregulatory Effect of Preventive Supplementation of Omega-3 Fatty Acid in a Complex Regional Pain Syndrome Type I Model in Mice

Paula Franson Fernandes  1 , Taynah de Oliveira Galassi  1 , Verônica Vargas Horewicz  1 , Afonso Shiguemi Inoue Salgado  2 , Josiel Mileno Mack  1   3   4 , Heloiza Dos Santos Baldança  1   5 , Ana Paula Ferreira da Silva  1 , Stephen Bruehl  6 , Edsel B Bittencourt  7 , Lynsey A Seim  8 , Daniel Fernandes Martins  1 , Franciane Bobinski  1
Affiliations

Immunoregulatory Effect of Preventive Supplementation of Omega-3 Fatty Acid in a Complex Regional Pain Syndrome Type I Model in Mice

Paula Franson Fernandes et al. Front Integr Neurosci. .

Abstract

Objective: Complex regional pain syndrome (CRPS) is usually triggered by trauma or a surgical procedure, and it typically becomes an established one after an intense inflammatory process with chronic pain and edema as the main symptoms. Available treatments for CRPS have low efficacy. This study aimed to evaluate the clinical and immunoregulatory effects of omega-3 polyunsaturated fatty acid (PUFA) supplementation on paw edema and anti- and pro-inflammatory cytokines and macrophage phenotypes in the chronic post-ischemia pain (CPIP) preclinical model of CRPS-Type I.

Methods: Female Swiss mice were supplemented with omega-3, corn oil, or saline and then submitted to the CPIP model of ischemia/reperfusion (I/R) injury. Supplementation was carried out for 30 days prior to and up to 2 or 15 days after the induction of CPIP, according to experimental protocols. The supplementation protocol included 1,500 mg/kg of omega-3 or corn oil through an intragastric route (gavage). Paw edema, interleukin- (IL-) 4, IL-10, transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor (TNF) were then measured in the paw skin and muscle by enzyme-linked immunosorbent assay (ELISA), and macrophage phenotypes (M1 and M2) assessed in the paw muscle by Western blotting.

Results: The CPIP model induced an increase in paw thickness up to 72 h post-I/R. Mice supplemented with omega-3 compared to the saline group displayed reduced edema but neither altered skin IL-4 or skin and muscle TGF-β1, TNF, and MCP-1 concentrations, nor did they exhibit significantly altered muscle macrophage phenotype on the 2nd-day post-CPIP. However, omega-3 supplementation reversed the I/R-related reduction in IL-4 in the paw muscle compared to groups supplemented with saline and corn oil. Furthermore, omega-3 promoted the reduction of IL-10 levels in the paw skin, compared to animals with lesions supplemented with saline, until the 2nd-day post-CPIP. On the 15th day post-CPIP, IL-10 was significantly increased in the muscle of animals supplemented with omega-3 compared to the saline group.

Conclusion: The results suggest that omega-3 PUFA supplementation has anti-inflammatory effects in the CPIP model of CRPS-Type I, significantly reducing paw edema and regulating concentrations of anti-inflammatory cytokines, including IL-4 and IL-10.

Keywords: complex regional pain syndrome; cytokines; edema; macrophages; omega-3.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Experimental design with chronic omega-3 supplementation for 30 days and then baseline assessment of paw thickness with a micrometer (μm) before the ischemia and reperfusion procedure. Subsequently, the CPIP animal model was induced. The omega-3 supplements continued daily up to the 4th day after the induction of the model when daily assessments of paw thickness were performed. Following 15 days, structures were removed for an ELISA biochemical analysis (A). In the second experiment, animals received the same supplementation protocol before the induction of the CPIP model and up to 2 days (48 h) after the induction of the model, where structures were removed for the biochemical ELISA and WB assays (B). CPIP, Chronic Post-Ischemia Pain; WB, Western Blotting; ELISA, Enzyme Immunosorbent Assay.
FIGURE 2
FIGURE 2
Effects of omega-3 supplementation on paw edema post-CPIP induction. Paw thickness evaluations of animals in Saline/Sham (n = 10), Saline/CPIP (n = 9), Omega-3/CPIP, and Corn oil/CPIP (n = 10) groups 1 day before the induction of the CPIP model (baseline, A), and time course of the saline, omega-3, or corn oil treatments on days 1, 2, 3, and 4 after model induction (A–D). Assessments at 24 (A) and 48 h (B) post-injury, and 1, 2, and 3 h post-treatment. Time course up to 2 h post-treatment, 72 h after the induction of CPIP (C). Paw thickness at 96 h post-CPIP and 1 h post-treatment (D). Data are expressed as mean ± SD compared using the two-way ANOVA with repeated measurements followed by Tukey’s test. #p < 0.05 and ###p < 0.0001 vs. Saline/Sham group; *p < 0.05 vs. Saline/CPIP group.
FIGURE 3
FIGURE 3
Effects of omega-3 supplementation on tumor necrosis factor (TNF) and monocyte chemotactic protein-1 (MCP-1) concentrations in the paw skin and muscle of mice 48 h after the induction of the CPIP model. TNF concentrations in the skin (A) and muscle (B) of Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. MCP-1 concentrations in the skin (C) and muscle (D) of Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. Data are expressed as mean ± SD of 6–7 animals per group, statistically assessed by the one-way ANOVA followed by Tukey’s test. #p < 0.05 vs. the Saline/Sham group.
FIGURE 4
FIGURE 4
Effects of omega-3 supplementation on the concentrations of interleukin- (IL-) 4, transforming growth factor-β1 (TGF-β1), and IL-10 in the skin and muscle of mice 48 h after the induction of the CPIP model. IL-4 concentrations in the skin (A) and muscle (B) of Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. TGF-β1 concentrations in the skin (C) and muscle (D) of Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. IL-10 concentrations in the skin (E) and muscle (F) of Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. Data are expressed as mean ± SD of 6–7 animals per group, statistically assessed by the one-way ANOVA followed by Tukey’s test. #p < 0.05 and ##p < 0.01 vs. the Saline/Sham group; *p < 0.05 and **p < 0.01 vs. the Saline/CPIP group. ıı p < 0.01 vs. the Omega-3/CPIP group.
FIGURE 5
FIGURE 5
Effects of omega-3 supplementation on the immunocontent of macrophages M1 markers: nitric oxide synthase 2 (NOS-2) (A) and CD86 (B), and M2 marker: Arginase-1 (C) on the mouse paw muscle 48 h after the model induction in Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. Data are expressed as mean ± SD of 6 animals per group, statistically assessed by the one-way ANOVA followed by Tukey’s test. ##p < 0.01 vs. the Saline/Sham group.
FIGURE 6
FIGURE 6
Interleukin-10 concentrations in the paw skin and muscle 15 days after the induction of the CPIP model. IL-10 concentrations in the skin (A) and muscle (B) of Saline/Sham, Saline/CPIP, Omega-3/CPIP, and Corn oil/CPIP groups. Data are expressed as mean ± SD of 6–7 animals per group, assessed by the one-way ANOVA followed by Tukey’s test. *p < 0.05 vs. the Saline/CPIP group.
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