This experimental, quantitative, randomized study was approved by the Ethics Committee for the Use of Animals of the State University of Western Paraná (protocol no. 13–22). All procedures were conducted at UNIOESTE (Cascavel campus) at the Laboratory for the Study of Injuries and Physiotherapeutic Resources and the Experimental Morphology Laboratory, in accordance with the ARRIVE guidelines 2.0 (Percie du Sert et al. 2020).

Obtaining animals and experimental design

Thirty-one male Wistar rats (3 months old at the beginning of the experimental protocol; mean body weight 340 g) were obtained from the UNIOESTE Central Animal Facility and acclimatized for 1 week. Animals were housed according to treatment groups, with turmeric-treated groups kept separately to avoid cross-contamination. Rats were randomly allocated into five groups: control (CTRL; n = 6), adjuvant-induced arthritis (AIA; n = 6), AIA + turmeric (AIA + T; n = 6), AIA + stair-climbing resistance exercise (AIA + E; n = 6), and AIA + turmeric + exercise (AIA + T+E; n = 7).

Sample size was estimated based on an ANOVA model (power 90%, α = 0.01) using G*Power 3.1.9.7 (Windows).

Adjuvant-induced arthritis

Complete Freund’s Adjuvant (CFA) (Gomes et al. 2014) was used to induce adjuvant-induced arthritis. Rats received 50 µL of CFA (0.5 mg/mL; Mycobacterium butyricum; Difco®) intradermally at the base of the tail. Control animals received saline (0.9% NaCl; Aster®). Seven days later, an intra-articular injection (50 µL CFA or saline for controls) was performed in the tibiofemoral joint of the right hindlimb under aseptic conditions using 1% iodized alcohol (Rialcool®).

For both injections, animals were gently restrained using a flannel cloth, as approved by the institutional Ethics Committee for the Use of Animals (protocol no. 13–22), and no anesthesia was used.

Animals were included in the arthritic groups only if visible tibiofemoral joint swelling was present 24 h after the intra-articular injection. Animals without joint swelling were excluded (n = 0).

Turmeric supplementation protocol

Turmeric supplementation was administered to AIA + T and AIA + T+E groups starting 24 h after intra-articular injection and continued for 15 days (Nonose et al. 2014). Animals received 100 mg/kg/day of Curcuma longa L. dry extract by gavage, with doses adjusted weekly according to body weight. CTRL, AIA, and AIA + E received water by gavage to control for handling stress.

Stair-climbing resistance exercise protocol

Exercise adaptation was performed before arthritis induction, consisting of 7 consecutive days of climbing (nine climbs per session, without load) to familiarize animals with the ladder apparatus (Jacob et al. 2022). The adaptation protocol was completed prior to the first CFA injection. The apparatus was a vertical wooden ladder (1.18 m high, 20.5 cm wide, 70° incline) with 67 iron rungs and a rest box at the top.

After adjuvant-induced arthritis was established, the resistance training period was initiated 24 h after the intra-articular injection and continued for 15 days, performed every other day. Each session consisted of three sets of eight climbs with progressive loads (25%, 35%, and 50% of body weight) using lead plates attached to the tail, with 2-min rest intervals between sets.

Functional assessments

All groups underwent joint swelling and grip strength assessments. During the adaptation week, animals were familiarized with the assessment equipment for 5 days. Measurements were performed blinded by the same researcher at the same time of day at five time points: baseline (BA; Day 1), after induction (FA.1; Day 11), and during follow-up (FA.2–FA.4; Days 15, 19, and 23).

Assessment of joint swelling

To assess joint swelling, the animals were restrained with a flannel for the duration of the measurement. Joint swelling was assessed by measuring the femoral–tibial joint diameter of the right hindlimb along the medial–lateral axis using a caliper (Mister®, Rio Grande do Sul, Brazil), according to Neves et al. (2020). Three measurements were obtained per animal, and the mean value was used (cm).

Grip strength assessment (muscle strength)

Grip strength was assessed in the right hindlimb using a grip strength meter (Insight®, Ribeirão Preto, São Paulo), according to Coradinia et al. (2015). The animal was gently pulled by the tail and trunk, allowing it to grasp the grid connected to the force transducer with the right hindlimb until it lost its grip. The peak force was recorded (g).

Euthanasia of the animals

On Day 23, at the end of the experimental period, the rats (4 months old) were weighed, measured (nasoanal length), and had the Lee index calculated. Euthanasia was performed via intraperitoneal injection of ketamine hydrochloride (240 mg/kg, Ketalar®, Brazil) and xylazine (45 mg/kg, Xilazin®, Brazil).

EDL muscle collection

The right extensor digitorum longus (EDL) muscle was collected for morphological and morphometric analyses. After removal of the tibialis anterior, the EDL was dissected, measured with a digital caliper (Digimess®, Brazil), and weighed on an analytical scale (Bel®, Brazil). Samples were divided for histological and histochemical analyses.

Histological studies of muscle fibers and connective tissue

The proximal segment of the right EDL was fixed in Metacarn for 24 h and stored in 70% ethanol. Samples were dehydrated in graded alcohol series, cleared in n-butyl alcohol, embedded in paraffin, and cross-sectioned (7 μm) using a Leipzig microtome.

Sections were stained with hematoxylin and eosin (Junqueira and Junqueira 1983). Muscle fiber nuclei, fiber area, largest and smallest diameters, and the capillary-to-fiber ratio were assessed in 10 fields per animal using a 40× objective. Capillaries were counted by two blinded assessors following Fernandes et al. (2012).

A histopathological index was applied (Zazula et al. 2022), considering lesion importance (w = 1–3) and extent (α = 0–6), with total score calculated as x = α*w (maximum = 320).

Connective tissue was evaluated using Masson’s trichrome staining (Bancroft and Stevens 1990). Ten microscopic fields per animal were analyzed using a 40× objective to quantify connective tissue as a percentage of total tissue area. For neuromuscular spindle analysis, all observable spindles in a section were counted under a 40× objective.

Morphological and morphometric study of the neuromuscular junctions

For neuromuscular junction evaluation, the distal segment of the right EDL was immersed in Karnovsky fixative (Karnovsky 1964) and stored refrigerated until processing. The muscle was longitudinally sectioned into 3–4 slices using stainless steel blades and subjected to the nonspecific esterase reaction (Lehrer and Ornstein 1959). Because this histochemical protocol requires free-hand thick sections, image acquisition has inherent limitations regarding focal depth. All images were acquired under identical optical conditions.

Neuromuscular morphometry (area, largest diameter, and smallest diameter) was obtained from 100 NMJs per animal from images acquired using a 20× objective.

Image acquisition and analysis

The morphological and morphometric analyses of the EDL muscle were based on images obtained by an Olympus DP71 camera (Tokyo, Japan) coupled to an Olympus Bx60® microscope with the aid of the DP Controller 3.2.1 276 program. The images were analyzed using Image Pro Plus 6.0® (Media Cybernetics, Maryland, USA).

Statistical analysis

The animal was considered the experimental unit (biological replicate), with 6–7 animals per group. For morphometric and histological analyses, multiple fields/structures were evaluated per animal and averaged to yield one value per animal for statistical comparisons.

Parametric body, morphometric, and histological data are presented as mean ± SD and were analyzed using Student’s t-test when comparing CTRL and AIA (to confirm arthritis induction).

Comparisons among arthritic groups (AIA, AIA + T, AIA + E, and AIA + T+E) were performed using two-way ANOVA followed by Fisher’s post hoc test, when appropriate.

Non-parametric data were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test.

Functional outcomes (joint swelling and grip strength) were analyzed using two-way repeated-measures ANOVA (time as within-subject factor and group as between-subject factor) and are presented as mean ± SEM, followed by Fisher’s post hoc test. Statistical significance was set at P < 0.05. Analyses were performed using XLStat 2014 (Addinsoft®, Paris, France).