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

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.

dissolved in DMSO (20%) for intra-peritoneal (i.p.) and intrathecal (i.t.) treatments and isotonic saline solution (NaCl 0,9%) (80%) immediately before use. Ptx i.p. treatment was conducted in two time periods, 30 min before and 12 h after the intense acute swimming session. This treatment protocol was based on the treatments used to inhibit TNF-a in DOMS (Borghi et al., 2014b). As the aim of this study was to demonstrate the proof-of-concept whether Ptx could potentially be repurposed for DOMS treatment, we first focused on verifying its activity using a known protocol that targets TNF-a in DOMS. I.t. injections were conducted in unconscious mice (targeting L4-L6 segment) under anesthesia with isoflurane (3% inhalation). I.t. treatment was performed only once to avoid local inflammation and enhancement of nociception (Almeida et al., 2000).
Animals were randomly divided in treatment groups and samples/analyzes were performed at indicated time points described in Fig. 1A. All parameters were previously standardized (Borghi et al., 2014a;Borghi et al., 2014b;Borghi et al., 2016;Borghi et al., 2021). All parameters mentioned were evaluated upon i.p. treatment with Ptx. The Ptx i.t. treatment was used only to demonstrate its spinal cord analgesic effect.

Intense acute swimming protocol to induce DOMS
Mice were placed in a glass box (45×28×25 cm, divided in six compartments) with approximately 20 liters of water at 31° ± 1°C as described previously (Borghi et al., 2014b). Briefly, each mouse was individually placed in one compartment and (Borghi et al., 2014b) swam all the same time during 120 min. After the intense acute swimming session or sham conditions, animals were dried and placed in cages together with their randomized respective group (Borghi et al., 2014a;Borghi et al., 2014b;Borghi et al., 2016;Borghi et al., 2021). This is not a stress protocol as we have already demonstrated (Borghi et al., 2014b).

Evaluation of muscle mechanical hyperalgesia
In a quiet room, mice were placed in acrylic cages (12×10×17 cm) with wire grid floors, 15-30 min before the start of the test. Evaluations consisted of evoking a hind paw flexion reflex with a hand-held force transducer (electronic von Frey anesthesiometer; Insight, Ribeirão Preto, São Paulo, Brazil) adapted with a 0.5 mm 2 contact area polypropylene tip.
We used the regular probe of electronic von Frey apparatus, since in a previous publication of our group (Borghi et al., 2014b) we demonstrated that using regular probe (0.5 mm 2 contact area) that elicits nociceptive responses per se, and large probe (4.15 mm 2 contact area) that does not elicit nociceptive responses per se, we obtained equivalent results in intense acute swimming-induced mechanical hyperalgesia, highlighting that the reason by our success in measuring muscle pain with a 0.5mm 2 diameter probe was due to intact cutaneous paw tissue added to simultaneous sensitized skeletal muscle tissue, the latter provoked by muscle overload induced by intense acute swimming DOMS model. During the investigations performed in the experiments, movement-elicited hyperalgesia was provoked by the pressure exerted by the von Frey probe on the plantar surface, which induces the dorsal flexion of the ankle joint (an antagonistic movement provided by the contraction of the soleus muscle), which leads to the passive stretch of the Achilles tendon, generating muscle distention. Thus, the end point of evaluations was always characterized by the removal of the paw followed by clear flinching movements when the muscle of the mice is distended (Borghi et al., 2014a;Borghi et al., 2014b). The measurements were performed only when the animals were not agitated, and with all four paws resting on the floor of the grid. Evaluations occurred between 6 to 48 h after the intense acute swimming session. We established the measurement timeline up to 48 hours in our acute swimming model, as we identified that the peak of pain-like behavior in the mouse in the present model occurs before that observed in humans (48-72 h), at the 24 th hour after the swimming exercise. During the pilot experiments that were conducted at the beginning of the project, significant pain was no longer observed after the 48 th hour. For this reason, we determined the cut-off of mechanical hyperalgesia evaluations at 48 h for mice. Eventually, exposition to a longer swimming period could achieve a model that lines up exactly with the human time course of DOMS. Two h of swimming is a considerable exercise duration. The Londrina State University Committee on Animal Welfare understood that this would be reasonable considering species differences, however, longer time periods of exercise would not be justifiable since a similar profile was found. Three repetitions of each measurement were used during the experiment to obtain the average of the final value. The results are expressed by delta (Δ) withdrawal threshold (in g) calculated by subtracting the mean measurements (indicated time points) after stimulus from the baseline measurements.
Behavioral analyses were carried out always by the same person, blinded to the treatments.

Evaluation of CK blood concentration
Blood samples were collected after the swimming session, and subsequently centrifuged (1.500 g, 4°C, 10 min), and the resultant plasma was assayed for CK levels according to the manufacturer's guideline (Ref: 117, Labtest Diagnóstico S.A., Lagoa Santa, MG, Brazil). The results were presented as creatine phosphokinase (U/L of plasma).

Rotarod performance test
The test is used to quantitate the effects of varied conditions and procedures upon motorplanning, with very high reliability (Rustay et al., 2003). The equipment is composed by a bar (2.5 cm diameter) separated into 4 compartments by disks of 25 cm in diameter (Ugo Basile, model 7600). The evaluations occurs when the bars rotate at a constant speed of 22 rotations (min). Mice were selected 24 h before the treatments by eliminating those that did not remain on the bar for two consecutive periods of 180 seconds. Selected mice were then evaluated at baseline, (0), 1.5, 3.5, and 5,5 h after the treatments with Veh or Ptx. The cutoff time used for al mice in the apparatus was 180 sec.

Evaluation of neutrophils recruitment to the muscle tissue
The intense acute swimming-induced neutrophil recruitment was evaluated at 24 h after the swimming session using MPO kinetic-colorimetric method and immunofluorescence assays (Borghi et al., 2014b;Ruiz-Miyazawa et al., 2018). MPO activity methods was already described (ref). For fluorescence analysis, the soleus muscle was collected after the LysM-eGFP mice were perfused through the ascending aorta with phosphate buffered saline (PBS) followed by 4% of paraformaldehyde (PFA) twice. Samples were subsequently fixed in PFA 4% for 24 h, and after this period, PFA was replaced by a solution of 30% saccharose and incubation for 3 additional days. After this, muscles were washed with PBS and embedded in optimum cutting temperature (O.C.T.) using Tissue-Tek ® reagent (Sakura ® Finetek USA, Torrance, CA). Sections of 10 micrometers (μm) were cut in a cryostat (CM1520, Leica Biosystem, Richmond, IL, USA) and processed for immunofluorescence (four samples per mice per slide/4 mice per group). For this experiment four samples (sections) per mouse per slide and 6 animals per group were applied. Four experimental groups were analyzed during the experiments, as follows, naïve, sham, vehicle-, and Ptx-treated groups. The representative images and quantitative analysis were created and performed using a confocal microscope (SP8, Leica Microsystems, Mannheim, Germany). Bright field channel was used simultaneously during acquisitions. Neutrophils count was quantitated in random fields by an experimenter blind to the treatments. The results were presented as neutrophils count per field (of muscle).

Analyses of oxidative stress parameters and gp91 phox mRNA expression by reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
Oxidative stress-related parameters (NBT reduction and GSH levels) were assessed in muscle tissue 4 h after the swimming session (Borghi et al., 2016). In addition to those oxidative stress tests, RT-qPCR assay was performed to determine the mRNA expression of gp91 phox as previously described (Borghi et al., 2016). Muscle samples were collected 4 h after the swimming session and homogenization in TRIzol™ Reagent (Thermo Fisher Scientific). Total RNA was isolated following the manufacturer's instructions. The purity of total RNA was measured by spectrophotometry, and the wavelength absorption ratio Nitroblue tetrazolium (NBT) reduction test was used for determining the production of superoxide anion. Samples were collected in 500 μL of saline solution, and 50 μL of the homogenate was transferred to a sterilized 96-well plate. Next, an addition of 100 μL of NBT solution (1 mg/mL) and incubation for 1 h at 37°C was conducted. The remining supernatant was then removed from plates, and the precipitated formazan in the wells was solubilized by adding 120 μL of 2M KOH and 140 μL of dimethylsulfoxide (DMSO).
The concentration of superoxide anion was evaluated by spectrophotometry via the reduction of the redox dye NBT at 600 nm (Multiskan GO Microplate Spectrophotometer, Thermo Fischer Scientific, Vantaa, Finland). The NBT reduction levels were corrected according to the total protein concentration. The results were presented as NBT reduction [optical density (OD)/mg of protein of muscle (Borghi et al., 2021). GSH muscle content assay was also determined using a spectrophotometric method (Borghi et al., 2014b).

ELISA Tests for cytokine production
Muscle and spinal cord (L4-L6) samples were collected immediately after (2 h) and 24 h after the swimming session, respectively, for the evaluation of TNF-a, IL-1b and IL-10 production by enzyme-linked immunosorbent assay (ELISA) using eBioscience kits (Affymetrix, San Diego, CA, USA). The technical procedure for collecting the lumbar segments, is related to the identification of the dorsal root ganglia, and then followed back the root to the spinal level using a stereo microscope. Samples were homogenized in appropriate buffer containing protease inhibitors and properly centrifuged for supernatant generation. During the assay, following initial blocking of the plates, they were coated overnight at 4°C with an immunoaffinity-purified polyclonal antibodies specific for evaluated cytokines. In the next day, recombinant murine diluted TNF-α, IL-1β and IL-10 standards and the samples were added in wells in duplicate and incubated by an additional period of 2 h at room temperature. Rabbit biotinylated immunoaffinity-purified antibodies anti-TNF-α, anti-IL-1β, anti-IL-10 were then added, followed by another incubation at room temperature with duration of 1 h. Subsequently, avidin-HRP was added to wells, and after an additional period of 30 min, the plates were properly washed and the color reagent o-phenylenediamine was added in the concentration of 200 µg per well. After these steps, reactions were blocked and measurements conducted spectrophotometrically at 450 nm (Multiskan GO Microplate Spectrophotometer, Thermo Fischer Scientific, Vantaa, Finland). The results were expressed as pg of each cytokine/100 mg of tissue (Borghi et al., 2015;Borghi et al., 2021).

Evaluation of spinal cord glial cells activation
The activation of spinal cord astrocytes and microglia was assessed by RT-qPCR and immunofluorescence techniques performed 24 h after the swimming session (Borghi et al., 2016;Borghi et al., 2021). For evaluation of GFAP, Iba-1, and CX3CR1 mRNA expressions, spinal cord samples were processed in the same way as that presented for

Statistical Analysis
Results are presented as means ± standard error mean (SEM) of assessments performed on 4 or 6 mice in per group during the experiment, depending on the method and are representative of two separated experiments. Two-way analysis of variance (ANOVA) was used to compare the groups and doses at all time points (curves). The analyzed factors were treatments, time, and time versus treatment interaction. When there was a significant time versus treatment interaction, one-way ANOVA followed by Tukey's t-test was performed for each time. Statistical differences were significant at P < 0.05. For detailed information on statistical analysis, please see Tables S1, which includes information about t test (P values), and one-or two-way ANOVA (F and P values).