Brief research report: Repurposing pentoxifylline to treat intense acute swimming-Induced delayed-onset muscle soreness in mice: Targeting peripheral and spinal cord nociceptive mechanisms

Abstract

In this study, we pursue determining the effect of pentoxifylline (Ptx) in delayed-onset muscle soreness (DOMS) triggered by exposing untrained mice to intense acute swimming exercise (120 min), which, to our knowledge, has not been investigated. Ptx treatment (1.5, 4.5, and 13.5 mg/kg; i.p., 30 min before and 12 h after the session) reduced intense acute swimming-induced mechanical hyperalgesia in a dose-dependent manner. The selected dose of Ptx (4.5 mg/kg) inhibited recruitment of neutrophils to the muscle tissue, oxidative stress, and both pro- and anti-inflammatory cytokine production in the soleus muscle and spinal cord. Furthermore, Ptx treatment also reduced spinal cord glial cell activation. In conclusion, Ptx reduces pain by targeting peripheral and spinal cord mechanisms of DOMS.

Keywords: cytokine; glial cells; muscle mechanical hyperalgesia; oxidative stress; pentoxifylline.

FIGURE 1

Experimental protocol and analgesic effect of pentoxifylline (Ptx). (A) Schematic representation of the experimental protocol used in the study. Ptx inhibits intense acute swimming–induced (B) muscle mechanical hyperalgesia and (C) increased plasmatic creatine phosphokinase (4.5 mg/kg dose; i.p.), and it does not induce (D) motor dysfunctions or impairments in (E) time spent in swimming behavior (the white color at the top of the bars indicates the immobility time). Results are presented as intensity of hyperalgesia (Δ reaction, in grams) and as creatine phosphokinase (total) (U/L of plasma) for CK plasmatic levels, and the results of motor and swimming performance are presented in seconds and minutes, respectively (n = 6 mice per group per experiment and is representative of two independent experiments). *p < .05 when compared with control groups, #p < .05 when compared with the Veh group (two-way ANOVA followed by Tukey’s post hoc).

FIGURE 2

Ptx reduces DOMS recruitment of neutrophils, oxidative stress, and cytokine production in the soleus muscle. Ptx (4.5 mg/kg dose; i.p.) inhibits intense acute swimming–induced (A) MPO activity, (B,C) LysM-eGFP⁺ neutrophil recruitment, (D) gp91 ^phox mRNA expression, (E) superoxide anion production, (F) GSH, (G) TNF-α, (H) IL-1β, and (I) IL-10 levels. Results are presented as the number of neutrophils ×10⁴ per milligram of muscle for MPO activity, neutrophil counts per field, muscle mRNA fold-change normalized to β-actin for gp91 ^phox expression, as NBT reduction (OD per milligram of protein of muscle for superoxide anion production), millimols per milligram of muscle for GSH levels, and picograms per 100 mg of muscle for cytokine levels (n = 6 mice per group per experiment and is representative of two independent experiments). Scale bar: 50 µm *p < .05 when compared with control groups, #p < .05 when compared with Veh group (one-way ANOVA followed by Tukey’s post hoc).

FIGURE 3

Ptx reduces cytokine production and glial cell activation in the spinal cord. Ptx (4.5 mg/kg dose; i.p.) inhibits intense acute swimming–induced (A) TNF-α, (B) IL-1β, and (C) IL-10 protein levels; (D) Gfap, (E) Iba1, and (F) Cx3cr1 mRNA expression; and GFAP (G,H) and (G,I) Iba-1 immunofluorescence intensity in the spinal cord. Boundaries of dorsal horn laminae (I–V) were delineated to better clarify the regions of staining. An illustration of the spinal cord in the right denotes the areas in which staining was quantified during the experiments. Results are presented as picograms per 100 mg of the spinal cord for cytokines, as the spinal cord mRNA fold-change normalized to β-actin for glial markers mRNA expression (n = 6 mice per group per experiment and is representative of two independent experiments), and as fluorescence intensity for glial protein expression (n = 4 mice per group per experiment and is representative of two independent experiments; scale bars: 100 µm for the inserts). *p < .05 when compared with control groups, #p < .05 when compared with Veh group (one-way ANOVA followed by Tukey’s post hoc).

FIGURE 4

Schematic representation of pentoxifylline modulatory mechanisms in experimental DOMS in mice. Ptx treatment reduced DOMS CK elevation, neutrophils recruitment, oxidative stress (increased gp91 ^phox mRNA expression and superoxide anion production and reductions in GSH levels), and cytokine production (TNF-α, IL-1β, and IL-10 levels) in the soleus muscle. In the spinal cord, Ptx treatment inhibited cytokine production (TNF-α, IL-1β, and IL-10 levels) and astrocytes and microglial activation at the mRNA (Gfap, Iba1, and Cx3cr1) and protein (GFAP and Iba-1) levels. These integrated peripheral and spinal mechanisms lead to the reduction of muscle pain (DOMS) by Ptx treatment.