Animals

Male DBA/1J mice were used. During experimentation, animals were maintained in a 12 h dark/light cycle, 20 to 24 °C temperature, and 40–60% humidity level. Water and food were administered ad libitum. All experiments were performed following the Guiding Principles for Research Involving Animals. This study was approved by the Research Ethics Committee of the Hospital de Clínicas de Porto Alegre (protocol number 18-0302). The study complies with the principles of the 3Rs: Replacement of animals by alternatives wherever possible, Reduction in the number of animals used, and Refinement of experimental conditions and procedures to minimize the harm to animals. All animals were anesthetized with isoflurane (Abbott, Abbott Park, IL, USA) during arthritis induction and euthanasia and were monitored twice a week to assess any behavioral change and loss of quality of life, observing signals of inactivity, suffering, stress, unbearable pain and no food or water intake.

Collagen-induced arthritis model

CIA was induced by 2 mg/ml of bovine type II collagen plus complete Freund’s adjuvant intradermal injection at the tail at day 0. At day 18, mice received a booster injection with bovine type II bovine collagen and incomplete Freund’s adjuvant. Animals were randomized into three groups: healthy mice without intervention (control group), arthritis-induced animals treated subcutaneously with PBS (vehicle group), and arthritis-induced animals treated with tofacitinib (tofacitinib group) at the dosage of 30/mg/kg/day (diluted in PBS 1X). The treatment with tofacitinib was subcutaneously administered, twice a day, from day 19 after CIA induction to day 45 (27 days of treatment) (Fig. 1). The dosing strategy was planned according to the literature [12] and the administration was decided in order to respect the short half-life of tofacitinib. Forty-five days after disease induction, animals were anesthetized with isoflurane and euthanized by cervical dislocation. Ankle joints were collected for confirmation of arthritis induction by histological analysis. In addition, serum samples were collected, and the muscles tibialis anterior (TA) and gastrocnemius (GA) were dissected, weighted, and processed for histological and molecular analyses, respectively.

Fig. 1

Experimental method during 45 days of disease induction

Clinical score

Animals were monitored daily during the whole experimental period by blinded evaluators, for clinical signs of arthritis according to a severity score: 0) without erythema and edema; 1) light edema and erythema; 2) mild edema and erythema; 3) severe edema and erythema in metatarsal and ankle joints; 4) all joints affected by edema and erythema and loss of function (ankylosis). Total animal severity scores were considered by the sum of each paw score, with being 16 the highest score [20]. The arthritis clinical score was measured by a blinded examiner.

Paw edema

Paw edema was measured in animals’ hind paws. This measure was performed by the submersion of animal paws in equipment called plethysmometer (Insight Ltda., Ribeirão Preto, Brazil). This equipment measures the dislocation of volume attributed to edema in animal paws [20]. Edema was measured by a blinded examiner.

Physical performance

A physical performance test was held to analyze the time of fatigue at days 0, 18, 25, 35, and 45 after CIA induction. Physical performance tests were done in the morning, at the same time period, and by the same evaluators. For the physical performance test, animals were initially placed on the treadmill for 5 min of ambiance before the test started. The treadmill started at 8.5 m/min speed and was raised by 2.5 m/min every 3 min reaching the maximum of 45 m/min. Animals were removed from the treadmill when exhausted, defined by the incapacity to keep running. Performance was estimated using two parameters: total duration of running (time in minutes) and distance traveled (meters). The distance was used as the main parameter to estimate resistance performance. Fatigue assay was performed by a blinded examiner.

Muscle strength

The maximum grip strength test was adapted from Deacon et al. [21]. Meshes with proper loads, each one amounting to 5, 20, 35, 50, 65, 80, and 95 grams were used. The mouse was held by the tail and suspended until it grasped the lighter weight with all paws for at least 3 recorded seconds. If the animal succeeded, it rested for the 30s before trying the next weight. If the animal failed three times with a 10s rest between each attempt, the longest time it was able to hold the weight was recorded. The following equation was used: Fmax = P3seg + (5*t < 3seg), where Fmax is the maximum calculated grip strength, “P3seg” is the heaviest load the animal held for 3 s, and “t < 3seg” is the longest time the animal held the heaviest load. The final result was expressed in grams (g) [22]. Grip strength assay was performed by a blinded examiner.

Nociception

Hind paw nociception was evaluated at days 0, 18, 25, 35, and 45 after CIA induction. Mice were placed individually in acrylic boxes. The experiment was performed by a blinded examiner, using an analgesimeter that presses the hind paw of mice and registers the intensity of the force applied when the animal withdraws its paw [23].

Joint histopathology

After anatomic dissection, joints were fixed in 10% formol for 72 h, decalcified in 10% nitric acid for 24 h, paraffin was embedded, sectioned, and stained with hematoxylin-eosin (HE). HE slides were analyzed by a blinded pathologist to establish the histopathological score [24].

Muscle weight and myofiber cross-sectional area

The TA and GA muscle weights were obtained after euthanasia and organ dissection. TA muscle was immersed in 10% buffered formalin for fixation for up to 2 days. The samples were dehydrated and paraffin-embedded. Slices 6 μm thick were arranged on microscope slides and HE stained using a standard protocol (Fischer et al. [25]). Myofiber cross-sectional area (CSA) measurement was determined using 10 pictures of each muscle sample, and 20 fibers of each picture were sized using the Image-Pro Express software (version 5.1.0.12, Media Cybernetics, Rockville, MD, USA) and counted by two blinded examiners.

Western blot analyses

Western blot analyses were performed as previously described [26]. Briefly, TA muscles were homogenized in a lysis buffer containing protease inhibitors (cat# A32955, Thermo Scientific) and phosphorylation protease inhibitors (cat# 4906845001, Roche). After quantification of protein concentrations using the Bradford method, the lysates or total proteins (25 μg/lane) were separated by SDS-PAGE on 12–16% gels and transferred onto polyvinylidene fluoride (PVDF) membrane (cat# IPFL00010, Millipore, Bedford, Mass). After being blocked with 5% milk, the membranes were probed with primary antibodies overnight at 4 °C. The primary antibodies used in this study were myogenin (1:1000, Sigma-Aldrich (SAB2501587, lot 9955P1)), MyoD (1:1000, Sigma-Aldrich (SAB4300397, lot 8715), Pax7 and GAPDH (1:1000, Sigma-Aldrich (G9545, lot 092M4800V)). Finally, the detection was made by the binding of the secondary antibody with the primary antibody, leading to a chemiluminescent signal. All raw data is available in the supplementary material file (Fig. 3).

Enzyme-linked immunosorbent assay

Serum samples were used for TNF-α (ELISA MAX Deluxe Set (430904) BioLegend, San Diego, CA, United States) and IL-6 (Mouse ELISA MAX Deluxe Set (431304), BioLegend, San Diego, CA, United States) detection by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions.

Statistical analysis

The sample size was calculated based on the previous study of our group, in which muscle atrophy was assessed in CIA mice by the myofiber CSA measurement area [27]. The authors found a 26 and 31% (p < 0.05) decrease of GA and TA myofiber CSA, respectively, in CIA group compared to CO group 45 days after disease induction. Therefore, considering this difference and using a power of 80% and a confidence of 95%, the sample size resulted in n of 9 animals per group. Considering the possibility of animal death due to error in the intradermal injection or the need for anticipation of death, 1 animal was added per group (rate 10%). So, our sample size was 10 mice per group. After confirmation of Gaussian distribution by Shapiro–Wilk and Kolmogorov–Smirnov tests, quantitative data were described as mean ± standard error of the mean (SEM). Comparison between groups was performed using one-way Analysis of variance (ANOVA) followed by Bonferroni’s test or Tukey’s test and two-way ANOVA followed by Bonferroni’s test or Tukey’s. All statistical tests were performed in GraphPad Prism software. Differences were considered statistically significant when p < 0.05.