Experiment 1: Development of OA flare model
Animals
Male C57BL/6 mice aged 7 weeks (18–20 g, Japan SLC, Hamamatsu, Japan) were housed under standard conditions. All experiments were carried out after 1 week of acclimatization to the environment. The experimental protocol was approved by the Institutional Review Board of our University for Animal Research (M-00078).
Preparation of MC
Bone marrow cells were isolated from the femurs of 8-week-old male C57BL/6 mice and cultured in RPMI 1640 medium containing 10% foetal bovine serum and 50% conditioned medium of WEHI-3 cells in plastic culture dishes [32]. The culture was continued for 5 weeks with weekly changes in the medium. Cell viability was determined by trypan blue staining. The concentration of the MC solution was then adjusted to 1 × 106 cells / 20 μl PBS [11].
Induction of OA and intra-articular injection of MC
OA was induced through a single intra-articular injection of monoiodoacetate (MIA), which is a well-established model associated with cartilage degeneration, joint destruction, and persistent pain behavior [31, 34, 46]. On day 0, the mice were anaesthetized by isoflurane inhalation. After sterilising their knee with 70% ethanol, MIA (0.5 mg) dissolved in 10 μl saline was injected into the right knee through the patellar tendon using a 30G Hamilton micro syringe. PBS was used as a control. On day 14, the MC solutions (1 × 106 cells / 20 μl PBS) were injected into the ipsilateral knee using the same procedure as above. The concentration of the MC solution was determined according to a previous intradermal injection of local MC injection intra-dermally [11]. PBS was injected as a control instead of MC solution. For subsequent analyses, mice were divided into four groups as follows according to the injected solutions on day 0 and day 14: OA flare (MIA + MC), OA (MIA + PBS), MC non-OA (PBS + MC), and PBS non-OA (PBS + PBS) (Fig. 1). First, two groups (MIA + MC and MIA + PBS) were created to investigate the role of MC infiltration in the OA joint. As no reports were available for MC injection into a normal joint, the MC non-OA (PBS + MC) group developed secondarily. The PBS non-OA (PBS + PBS) group was used as control.
Histopathological analysis
Mice were euthanized on day 22, and their knees were extracted and fixed in 4% paraformaldehyde for one day, decalcified in 13% formic acid solution for 2 weeks, and then embedded in paraffin. The specimens were sagittally sectioned at 5 μm thickness and stained with toluidine blue. Histological sections were observed using a Nikon ECLIPSE 80i microscope (Nikon, Tokyo, Japan). Histopathologic classification of the severity of OA was graded using a modified Mankin score ranging from 0 to 13 points (0 as best, 13 as worst) [47]. In addition, MCs were identified as cells with round or elliptical morphology and numerous cytoplasmic metachromatic granules [36] at the meniscosynovial junction. The number of MCs in the meniscosynovial junction was counted at 400 × magnification. The area of the meniscosynovial junction was then measured using image analysis software (Image J) [37] and the number of MCs was normalised by the area (number/ mm2).
Immunofluorescence staining
Immunofluorescence staining was performed to confirm the distribution of injected (exogenous) MC over time. Green fluorescent protein (GFP) expressing MCs were obtained from C57BL/6-Tg (CAG-EGFP) mice provided by Japan SLC (Hamamatsu, Japan) cultured for 5 weeks, and they were injected into the OA knees (MIA injected 14 days ago) using the same protocol as above. Mice were euthanized at 1, 3, and 7 days after injection of GFP-expressing MCs, and paraffin-embedded sections of the knee were obtained at each point in time. The presence of MCs, including both exogenous and endogenous cells, was investigated using mast cell protease-6 (MCP-6) immunofluorescence staining. The knees were extracted and fixed in 4% paraformaldehyde for one day, decalcified with 0.5 M EDTA for 2 weeks, and then embedded in paraffin. Following deparaffinization in xylene and washing with graded alcohol, activation with 0.1% trypsin was performed at 37 °C for 30 min. After blocking with 5% normal goat serum for GFP and 5% normal donkey serum for MCP-6, the slides were incubated at room temperature (20–25 °C) for an hour. Primary antibodies against rabbit GFP (1:500, Novus Biologicals, CO, USA) and goat MCP-6 (1:500, Santa Cruz Biotechnology, TX, USA) were applied to the sections and incubated overnight at 4° C. The next day, sections were incubated with the secondary antibody for 2 h. Goat anti-rabbit IgG Alexa Fluor 546 (1:400, Thermo Fisher Scientific, MA, USA) and donkey anti-goat IgG Alexa Fluor 488 (1:400, Thermo Fisher Scientific, MA, USA) were used as the corresponding secondary antibodies. Before, between, and after each incubation step, the sections were washed three times for 5 min PBS. Finally, all sections were mounted with Vectashield (Vector, Burlingame, CA). Sections were viewed with an FV-1000D laser confocal microscope (Olympus, Tokyo, Japan) and GFP/MCP6 immuno-reactive cells were observed.
Pain behavior test
Mice were individually placed in a transparent container at room temperature, and their spontaneous activities were continuously recorded using a digital camera (Nikon, Tokyo, Japan) for 10 min. Two observers independently watched the recordings and provided a semi-quantitative evaluation of pain behavior using Stance score [12] as follows: 0 = No visible impairment of gait or stance, the foot firmly placed flat on the surface with normal spread of toes. 1 = Moderate impairment of stance, the foot is placed on the ground with toes tightly contracted. 2 = Severe impairment of gait and stance; foot either entirely elevated from the ground or only the lateral part of the foot lightly touching the ground and toes tightly pulled together. As the ‘stance’ varied frequently during the observation period, the highest score maintained for at least 10 s was therefore assigned as the final score. Intermediate scores (0.5 and 1.5) were used for animals that displayed a behavior in between the definitions described above. Scoring was performed on days 0, 7, 14, 14 + 3 h (3 h after injection), 15, and 22 (Fig. 1). The average score from the two observers was used for data analyses. Ipsilateral paw hyperalgesia was assessed simultaneously. After 30 min of acclimatisation, paw withdrawal threshold was evaluated using von Frey filaments (North Coast Medical Inc., Morgan Hill, CA, USA) 1.65, 2.36, 2.44, 2.83, 3.22, 3.61, 3.84, 4.08, 4.17, and 4,31 (corresponding to 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, and 2.0 g, respectively).
Real time-polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated from the right side of the infrapatellar fat pad on day 22, and mRNA levels of IL-1β, TNF-α, IL-6, ADAMTS-4, and NGF was measured by the quantitative RT-PCR intercalated method. cDNA was synthesised using the ReverTra Ace® qPCR RT Kit (Toyobo, Osaka, Japan). Reaction mixtures for RT-PCR were composed of 2 μl cDNA, 1.6 μM specific primer pair, 10 μl TB Green Premix Ex Taq II (Takara, Kyoto, Japan), 0.4 μl ROX reference dye, and 6 μl nuclease free water in a final volume of 20 μl. Quantitative PCR was performed using a real-time PCR system (StepOnePlus; Thermo Fisher Scientific, MA, USA) to detect the relative mRNA expression levels. The PCR cycle protocol was as follows: the holding stage performed one cycle at 95 °C for 30 s, the cycling stage was repeated 40 times at 95 °C for 15 s, and at 60 °C for 60 s. mRNA expression was normalised to the levels of HPRT mRNA.
Experiment 2: Treatment of OA flare model with tranilast
To investigate the possibility of pharmacological suppression of MC-injection-induced acute flare, tranilast, an established MC stabilizer drug, was systemically administered to OA flare mice. This is according to a previous report using a collagen-induced arthritis model mimicking RA [14]. Tranilast has been used therapeutically for many years as an anti-allergic drug against bronchial asthma, allergic rhinitis, atopic dermatitis, and hypertrophic scars [9], and has been approved by the National Insurance System in our country. In the OA flare group, 1 ml of tranilast (400 mg/kg/day) or PBS was injected intra-peritoneally with a 27G syringe once a day from day 15 to 21. According to previous studies, tranilast was administered orally [40] or through an intraperitoneal injection [14], yet no study has been conducted using a local injection. In this study, we selected intraperitoneal injections to avoid differences in tranilast intake by oral administration. The mice were divided into two groups depending on the tranilast injection: the T ( +) and T (-) groups. To evaluate the pharmacological effects of tranilast, histology (modified Mankin score and number of MCs), pain behavior (stance score), and RT-PCR (IL-1β, TNF-α, IL-6, ADAMTS-4, and NGF) were examined in an identical manner to the first experiment. An overview of the experimental protocol is presented in Fig. 1.
Statistical analysis
The primary outcome of this study was histological changes in the OA flare model compared with the OA model in Experiment 1, and the pharmacological inhibitory effects of tranilast against this altered histology in Experiment 2. Pain behavior and joint inflammation were set as secondary outcomes in both experiments. Our sample size was determined by referring to a previous study that examined the modified Mankin score in a mouse MIA OA model. In the study, it was found that a 20% decrease in this histological score after a specific treatment was statistically significant [1]. Calculations were performed according to this difference with 90% statistical power and α level of 0.05, and it was determined that at least 10 mice were needed for OA flare and OA group in Experiment 1 and, T ( +) and T (-) group in Experiment 2. Finally, a sample size of 13 mice were chosen to allow for possible dropouts. Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni correction was used to compare pain behavior, modified Mankin score, and MC number among the four groups (OA flare, OA, MC non-OA, and PBS non-OA). The Mann–Whitney U test was used to compare pain behavior, modified Mankin score, and MC number with or without tranilast administration to the OA flare mice (T ( +) vs. T (-) groups). To evaluate mRNA gene expression levels between the groups (OA flare vs. OA group, and T ( +) vs. T (-) group), the Mann–Whitney U test was also used. Statistical significance was set at p < 0.05. All statistical analyses were performed using SPSS software (ver. 26.0; SPSS, Chicago, IL, USA).