2.3. Carrageenan-Induced Inflammation and Treatment Administration

Carrageenan (CAR)-induced inflammation is a recognized and highly reproducible model of acute inflammation and inflammatory pain [39,40]. Briefly, rats were anesthetized with 5.0% isoflurane in 100% O2 (Baxter International, Rome, Italy) and received a subplantar injection of carrageenan (CAR, 0.1 ml/rat of a 1% suspension in saline) (Sigma-Aldrich, Milan, Italy) with a 27-gauge needle into the right hind paw, as previously described [40,41]. Every compound was dissolved in carboxymethylcellulose (1% w/v in NaCl solution) and rats were randomly allocated to one of four groups and treated with

CAR + curcumin (10 mg/kg)

CAR + micronized PGA (20 mg/kg)

CAR + co-micronized PGA-Cur (2:1) (30 mg/kg)

CAR + carboxymethylcellulose (vehicle group)

As a sham group, saline was injected instead of CAR (control group). Each study compound (or vehicle) was administered orally by gavage as a single administration 30 min before CAR injection (N = 6 animals/group). Doses were chosen based on a dose–response study carried out in our lab and previous results [30]. The animals were sacrificed at 6h post CAR injection by isoflurane overdose. All analyses were performed in a blinded manner (i.e., the operator who assessed the below detailed parameters and the statistician who analyzed the data were both unaware of the treatment group).

Paw volume (mL) was measured using a plethysmometer (Ugo Basile, Varese, Italy) immediately prior to CAR injection and thereafter at 30 min and hourly intervals for 6 h. Edema was expressed as the increase of paw volume at each time point relative to pre-injection value [30]. Results are reported as paw-volume change (mL).

The hyperalgesic response to heat was determined at different time points (0, 30 min, and hourly intervals for 6 h) based on the method described by Hargreaves et al. [42], using a Basile Plantar Test (Ugo Basile, plantar test apparatus 7371; power requirement: 230–115 V, 60–50 Hz, 0.6 A maximum) as previously described [41]. Results are expressed as paw withdrawal latency changes (seconds).

Histological analysis of hematoxylin and eosin (H/E)-stained paw tissue was performed as previously described [41]. Briefly, the degree of tissue inflammation was evaluated on a 6-point score, from 0 (no inflammation) to 5 (severe inflammation) [43]. The photographs obtained (N = 5 photos from five slides for each sample) were collected from all animals in each experimental group. The histological coloration (five slides for each same sample) was repeated three times on different days.

Myeloperoxidase (MPO) activity, an index of neutrophilic granulocyte infiltration, was evaluated as previously described [30,41]. Briefly, the rate of change of the absorbance was measured spectrophotometrically at 650 nm. MPO activity was measured as the quantity of enzyme degrading 1 mM of peroxide/min at 37 °C and expressed as units per gram of wet tissue weight.