This study was performed on Sprague–Dawley male rats (200–230 g, 7 weeks old, Envigo RMS Srl, S. Pietro al Natisone, Udine, Italy). Ten rats were used for each treatment group (see below). Food and water were available ad libitum. The University of Messina Review Board for the care and use of animals authorized the study. Animal care was in accordance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with EEC regulations (O.J. of E.C. L 358/1 12/18/1986).
Co-ultramicronized PEA-Q was kindly provided by Epitech group SpA (Saccolongo, Italy). PEA-Q is the result of the joint ultramicronization - by jet-milling technology - of a mixture made of PEA and quercetin in a 5:1 ratio by mass. All other compounds were obtained from Sigma-Aldrich, Milan, Italy. All chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl, Baxter International, Rome, Italy).
Rats were anesthetized with 5.0% isoflurane in 100% O2 (Baxter International, Rome, Italy) and received a subplantar injection of CAR (0.1 ml/rat of a 1% suspension in saline) (Sigma–Aldrich, Milan, Italy) as previously reported . Injections were performed using a 27-gauge needle inserted into the pad region of the glabrous skin on the underside of the right hind paw. At 6 h following CAR injection, rats were sacrificed by anaesthetic (isoflurane) overdose.
All analyses conducted in CAR-injected rats (see details below) were performed in a blinded manner.
MIA induction model
OA was induced by intra-articular injection of MIA in the right knee joint as previously described . On day 0, rats were anesthetized with 5.0% isoflurane in 100% O2. A volume of 25 μl saline +3 mg of MIA was injected into the knee joint through the right intrapatellar ligament. The left knee received an equal volume of saline. MIA was prepared in sterile conditions and injected using a 50 μl Hamilton syringe with a 27 gauge needle that was inserted into the joint for about 2–3 mm. On day 21 post-MIA injection, rats were sacrificed by anaesthetic overdose and perfused with 4% paraformaldehyde. All analyses conducted in MIA-injected rats (see details below) were performed in a blinded manner.
The experiment was divided in two steps. First, we conducted a preliminary study to evaluate the effect of PEA-Q on inflammation and pain-related events (i.e., thermal hyperalgesia) in the CAR-induced paw oedema model. Rats were arbitrarily allocated to the following treatment groups, each compound being administered orally 30 min before CAR injection:
CAR + vehicle (saline): rats were subjected to CAR-induced paw oedema, as described above (N = 10);
CAR + quercetin: same as the CAR + vehicle group plus 3.3 mg/kg quercetin dissolved in carboxymethylcellulose (1.5% w/v in saline) (N = 10);
CAR + PEA-Q (10): same as the CAR + vehicle group plus 10 mg/kg PEA-Q dissolved in carboxymethylcellulose (1.5% w/v in saline) (N = 10);
CAR + PEA-Q (20): same as the CAR + vehicle group plus 20 mg/kg PEA-Q dissolved in carboxymethylcellulose (1.5% w/v in saline) (N = 10);
CAR + meloxicam: same as the CAR + vehicle group plus 0.2 mg/kg meloxicam dissolved in saline (N = 10).
sham group: same surgical procedure as the CAR group, except that saline was administered instead of CAR (N = 10).
Doses were chosen based on a dose-response studied carried out in our lab and on existing literature data. The dose of quercetin used is equivalent to 20 mg/kg PEA-Q. Based on preliminary results, we evaluated the anti-inflammatory and analgesic effects of PEA-Q in an experimental model of MIA-induced OA pain (definitive study). Rats were randomly divided into the following treatment groups, each compound being administered orally three times per week for 21 days, starting the third day after MIA injection:
MIA + vehicle (saline): rats were subjected to induction of OA pain as described above (N = 10).
MIA + PEA-Q (10): same as the MIA + vehicle group plus 10 mg/kg PEA-Q dissolved in carboxymethylcellulose (1.5% w/v in saline) (N = 10);
MIA + PEA-Q (20): same as the MIA + vehicle group plus 20 mg/kg PEA-Q dissolved in carboxymethylcellulose (1.5% w/v in saline) (N = 10);
MIA + meloxicam: same as the MIA + vehicle group plus 0.2 mg/kg meloxicam dissolved in carboxymethylcellulose (1.5% w/v in saline) (N = 10);
sham-operated group: rats received an intra-articular injection of saline (25 μl) instead of MIA (N = 10).
Quercetin was not tested in this set of experiments, given the lack of effect observed in the previous set. Doses were chosen based on a dose-response study carried out in our lab and on existing literature data. The timing of oral administration was based on a previous study .
The effect of PEA alone was not investigated, since oral administration of micronized and ultramicronized PEA had been shown to reduce inflammation and pain in several experimental models, the effective dose being 10 mg/kg [13, 14], thus higher than the dose equivalent to 10 mg/kg PEA-Q used here. Moreover, oral administration of naïve PEA (non-micronized) did not exert any significant effect against inflammation and hyperalgesia up to 10 mg/kg .
Assessment of CAR-induced paw oedema
Oedma was assessed by directly measuring changes in paw volume using a plethysmometer (Ugo Basile, Varese, Italy), as previously described . Paw volume was measured immediately prior to CAR injection and thereafter at hourly intervals for 6 h. Oedema was expressed as the increase in paw volume (ml) after CAR injection relative to pre-injection value. Six hours after CAR injection, percentage inhibition (protection) against oedema formation was calculated as follows and taken as an index of anti-inflammatory activity:
Percentage inhibition of inflammation = [(Vc-Vt)/Vc] × 100, where;
Vc = mean paw oedema volume in the control group at 6 h;
Vt = mean paw oedema volume in the drug-treated group at 6 h.
Pain-related behavioral analysis in the CAR model
The hyperalgesic response to heat was measured at different time points (0, 30 min, 1, 2, 3, 4 and 5 h) based on the method described by Hargreaves et al.  using Plantar Test 7371 (Ugo Basile, Italy). Briefly, animals were allowed to move freely within an open-topped transparent plastic chamber. After an acclimation period, a mobile infrared (I.R.) radiant heating source (IR 60) was placed under the glass floor and focused onto the hind paw. When the animal felt pain and withdrew its paw, the I.R. source switched off and the reaction time counter stopped. The withdrawal latency to the nearest 0.1 s was then automatically determined and recorded. A cutoff time of 20 s was set, i.e., if the rat failed to respond by 20 s the test was stopped in order to prevent tissue damage in non-responsive animals. Each point represents the delta change (sec) in withdrawal latency (withdrawal latency of contralateral minus withdrawal latency of injected paw) at each time point. Results are expressed as paw withdrawal latency changes (sec).
Percentage anti-hyperalgesic activity 5 h after CAR injection was calculated as follows.
Percentage anti-hyperalgesic activity = [(Vt-Vc)/Vt] × 100 where:
Vc = mean paw withdrawal latency in the control group at 5 h;
Vt = mean paw withdrawal latency in the drug-treated group at 5 h.
Histological analysis following CAR injection
Six hours after intraplantar CAR injection, animals were terminally anesthetized and paw biopsies collected. Histological analysis of haematoxylin and eosin-stained paw tissue was performed as previously described . The degree of paw damage was evaluated according to Bang and Coll. , on a six-point score: 0 = no inflammation, 1 = mild inflammation, 2 = mild/moderate inflammation, 3 = moderate inflammation, 4 = moderate/severe inflammation and 5 = severe inflammation. The photographs obtained (n = 5 photos from five slides for each sample) were collected from all animals in each experimental group. The histological coloration (5 slides for each same sample) was repeated three times on different days.
Myeloperoxidase (MPO) activity following CAR injection
MPO activity, an index of neutrophilic granulocyte infiltration, was evaluated as previously described . Briefly, after animals were terminally anesthetized paw tissues were collected and homogenized in a solution containing 0.5% hexadecyltrimethylammoniumbromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20000 g (4 °C). The supernatant was allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was measured as the quantity of enzyme degrading 1 mM of peroxide min−1 at 37 °C, and expressed in units per gram of wet tissue weight.
Assessment of MIA-induced mechanical allodynia
Mechanical allodynia was evaluated using a dynamic plantar Von Frey hair aesthesiometer on day 0 and 3, 7, 14 and 21 days post-injection (Ugo Basile, Comerio, Italy). Rats were located on a metal mesh surface in a chamber in a room with controlled temperature (21-22o C) and were allowed to adapt for 15 min before testing began. The touch stimulator part was positioned under the rat. When the aesthesiometer was activated, a plastic monofilament touched the paw in the metatarsal region. The filament exercised a gradually increasing force on the plantar surface, starting below the threshold of detection and increasing until the stimulus became painful and the rat removed its paw. The force required to produce a paw withdrawal reflex was recorded automatically and measured in grams. A maximum force of 50 g and a ramp speed of 20 s were used for all the aesthesiometry tests. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were calculated.
Motor function analysis (walking track analysis) following MIA injection
Motor functional recovery of the rear limb was evaluated by walking track analysis, a reliable and easily quantifiable noninvasive method based on gait analysis by means of specific footprint parameters . In brief, rats were previously trained to walk down a track with a dark end, covered with strips of white paper. Tracks were obtained by wetting the rat’s hind feet with water soluble black ink. Walking track analysis was performed in all animal groups before MIA injection (day 0) and 3, 7, 14 and 21 days post-injection. From the footprints, several measurements are taken between different anatomic landmarks (e.g., width and length of the footprint) and then incorporated in a mathematical formula, allowing the calculation of the functionality index of the sciatic nerve (SFI), with values close to 0 indicating normal function, and values tending to −100 indicating total impairment .
Histological analysis of MIA-injected rats
On day 21 post-MIA injection, rats were sacrificed and perfused with 4% paraformaldehyde. The MIA- and vehicle-injected tibiofemoral joints were dissected and post-fixed in neutral buffered formalin (containing 4% formaldehyde), decalcified in EDTA and processed as previously described. After decalcification, the specimens were embedded in paraffin. Mid-coronal tissue sections (5 μm) were stained for evaluation; all histomorphometric analyses were performed by an observer blinded to the treatment group. Sections were stained with haematoxylin and eosin and observed by light microscopy (Dialux 22 Leitz; Leica Microsystems SpA, Milan, Italy). Histopathological analysis of the cartilage was assessed by the modified Mankin score . Briefly, the score assessed: (i) structure, (ii) cellular abnormalities, and (iii) matrix staining of cartilage sections and ranged from 0 (= normal histology) to 12 (= complete disorganization and hypocellularity). The photographs obtained (n = 5 photos from five slides for each sample) were collected from all animals in each experimental group. The histological coloration (5 slides for each same sample) was repeated three times on different days.
Serum concentration of inflammatory, nociceptive and matrix degradation markers following MIA injection
The concentration of tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), nerve growth factor (NGF), and matrix metalloproteinase-1-3-9 (MMP-1, MMP-3, MMP-9) were measured in serum using commercial colorimetric ELISA kits (TNF-α, IL-1β, NGF: Thermo Fisher Scientific, DBA s.r.l. Milan Italy; MMP-1 MMP-3 MMP-9: Cusabio, DBA s.r.l. Milan Italy).
Data are expressed as mean ± standard error of the mean (SEM) of N observations, N representing the number of animals analyzed, with the exception of the ordinal level variable (i.e., histological score), for which median and range were used. In experiments involving histology, the figures are representative of at least three experiments performed on different days. The response over time was analyzed using a generalized linear mixed model (GLMM) for repeated measures, followed by Tukey-Kramer post-hoc analysis. One time evaluations with continuous level data were analyzed using ANOVA followed by Bonferroni-Holm post hoc analysis. Kruskal-Wallis test followed by Dunn’s test for post-hoc comparisons with Bonferroni-Holm p correction was used for the histological score, due the ordinal level nature of the variable (i.e., 0 to 5 point scale). Data were analyzed using SAS v9.2 (SAS Institute, Cary, NC, USA). The significance threshold was set at 0.05. Exact p values are reported, unless less than 1 out of 10,000 (reported as p < 0.0001), 0.0001 being the lower limit for the statistical program.