Animals

Male Wistar rats (200–250 g) aged 8 ± 1 weeks were used in this study. The animals were kept at constant room temperature (22 ± 2 °C) and maintained in a 12-h light/dark cycle. All animals were fed ad libitum with standard feed and water. All experimental procedures were carried out according to a protocol approved by local animal bioethics Committee of Instituto Nacional de Psiquiatría Ramón de la Fuente (NC123280.0 and NC17073.0), national [Norma Oficial Mexicana (NOM-062-ZOO-1999)], and international regulations (Zimmermann 1983) to care and use of animal in experimental pain. The rats were manipulated for adaptation to space and experimental conditions at least 3 days before testing.

Preparation and analysis of the aqueous extract of Brassica oleracea var. italica sprouts (AEBS)

Seeds of broccoli (Brassica oleracea var. italica) untreated and ready for sprouting were purchased from Intersemillas S.A. (Valencia Spain). The seeds were washed with 0.5% sodium hypochlorite solution for 2 h and rinsed with Milli-Q type water, followed by aeration for 24 h. The washed seeds were then germinated in a dark chamber at 28 °C for two days, and the germinates were transferred to a growth chamber under controlled conditions (16 h/8 h light/ darkness; 25 °C/ 20 °C air temperature, 60%/80% relative humidity), for six days. Then, sprouts were harvested and freeze-dried (Baenas et al. 2014). The AEBS was prepared as follows: The powder (1.6 g) was resuspended in 50 mL of Milli-Q water and macerated overnight at room temperature. Then, the sample was centrifuged, and the supernatant was filtered and freeze-dried (Baenas et al. 2017). Following the same protocol of Baenas et al. (2017), we analyzed the composition of the broccoli sprouts extract (AEBS) (Table 1).

Table 1 Quantification of sulforaphane, glucosinolates and phenolic compounds (mg g-1 d.w.) in broccoli (Brassica oleracea var. italica) sprouts and the aqueous extract (AEBS)Reagents and drugs

SFN (Cayman Chemical Company Inc. USA), tramadol (TR, AMSA Laboratorios, Mexico), ketorolac (KET, Liomont, S.A. de C.V. Mexico), and indomethacin (INDO, Sigma-Aldrich, St. Louis, MO. USA) were used in this study. All these drugs were prepared with the vehicle (saline solution, 0.9% NaCl). The 1% λ carrageenan (Sigma-Aldrich, St. Louis, MO. USA) was dissolved in saline solution to induce oedema. Oedema was analyzed with Masson’s trichrome stain kit (Hycel®).

Experimental protocol

Different doses of the AEBS using enteral (1000 and 2000 mg/kg, p.o.) or parenteral (30, 100, and 300 mg/kg, i.p.) administration, and a single dosage of SFN (0.1 mg/kg, i.p.), as well as the reference drugs INDO (20 mg/kg, p.o.), KET (20 mg/kg, i.p.), or TR (50 mg/kg, i.p.), were compared to the vehicle groups (p.o. or i.p.) in the nociceptive and inflammatory pain models of the plantar test and the carrageenan-induced oedema test, respectively. All the treatments were explored after 30 min of their administration (Guadarrama-Enríquez et al. 2018).

Thermal nociception test

In a preliminary study (Baenas et al. 2017), it was observed that AEBS produced antinociceptive effects in both neurogenic and inflammatory phases of the formalin test. Since the first phase of this test is considered to involve central antinociceptive mechanisms, sixty-six rats were divided into eleven groups of 6 rats per group to receive different doses of the AEBS (30, 100, and 300 mg/kg, i.p. or 1000 and 2000 mg/kg, p.o.) or SFN (0.1 mg/kg, i.p.) and compared to the vehicle groups (i.p. or p.o., respectively) or the reference analgesic drugs INDO 20, p.o., KET 20 or TR 50, i.p., explored in the plantar test. A previous exploratory activity was also tested using the open field test to rule out the possible sedative effects already known for analgesic drugs acting at central nervous system (CNS) as described below.

Open-field test. –After 30 min of treatment, the sedative-like response was evaluated by registering ambulatory activity of rats in the open field test. For this, ambulatory activity was tested by placing an individual rat in an acrylic box divided into twelve squares (9 cm × 9 cm). During the experiment, the number of squares explored by each animal with their four limbs was counted during 2 min (Baenas et al. 2017).

Plantar test.—Previously to the nociceptive test, rats were acclimated in Plexiglas chambers with heated glass floor for 30 min. Immediately after sedative-like response evaluation, rats were evaluated in the Hargreaves’ apparatus for thermal hyperalgesia. A radiant heat source with a locator light (intensity 60 Hz) was focused under the plantar surface of the right hind paw of the rat until produce a response of shaking or licking. Time of cut of this test was 20 s to prevent tissue damage. The latency of paw withdrawal was determined from an average of 3 separate trails, taken at 3-min interval. Data are expressed as the maximal possible effect (MPE%) calculated as follows (Hargreaves et al 1988; González-Ramírez et al. 2012; Cheah et al. 2017):

$$\mathrm\%=\frac\,}-\mathrm\, }}-\mathrm\, }}\times 100$$

Carrageenan-induced rat paw oedema

In a previous study (Baenas et al. 2017), the antinociceptive response of AEBS was reported in a second phase of the formalin test suggesting anti-inflammatory activity. Hence, this effect was reinforced by evaluating antiedema activity in vivo using sixty rats which were divided into ten groups of 6 rats per group to receive different doses of the AEBS (30, 100, and 300 mg/kg, i.p. or 1000 and 2000 mg/kg, p.o.) or SFN (0.1 mg/kg, i.p.) and compared to the vehicle groups (i.p. or p.o., respectively) or the reference analgesic drugs INDO 20, p.o., or TR 50, i.p., and tissular damage by histological analysis in this study. The oedema was analyzed in vivo using a vernier caliper, while a histological analysis of the paws of rats, after being euthanized using a CO2 chamber, was performance. Stomachs of the rats were also dissected to explore possible gastric damage as it was observed in the presence of NSAIDs like reference drugs.

Thirty minutes after treatment administration, plantar oedema was induced in the subplantar region of the right hind paw of rats with 50 μL 1% λ carrageenan. Inflammation was determined by measuring the thickness of oedema with a vernier caliper at times 0, 1, 2, 3, 4, 5, 6, and 24 h (González et al. 2007). The values were expressed as % of inflammation in the absence or in the presence of the several treatments calculated using the following formula:

$$\mathrm= \frac\,}-\mathrm\,}}\,}}\times 100$$

Histology.—Animals showing significant reduction of oedema in the first 6 h were euthanized, and their hind paws were used for histological analysis. Skin, tarsus, metatarsal, and phalanx bones of paws were removed to obtain only muscle and connective tissues. Tissues were embedded in 30% sucrose at 4 °C for 72 h and after in TissueTek® for 72 h to slice. Tissues were cooled, put-on longitudinal position, and trimmed to obtain slices (thickness 10 μm). Then, tissues were treated with Masson’s trichrome stain (Hycel®). They were then mounted on the gelatin microscope slides in the mounting medium. All tissue samples were representative of 4 animals.

Gastric damage.—At the end of the test, all animals were euthanized into the CO2 chamber, and the stomachs were dissected to examine presence of possible gastric lesions as previously described (Baenas et al. 2017).

Acute toxicity

To determine the median lethal dose (LD50), the AEBS was given to rats using a preliminary maximal dosage of 2000 mg/kg, by i.p. or p.o. route of administration, according to the OECD guideline for testing of chemicals (2001). After treatment, rats were kept under observation for 14 days to register behavioral changes on ambulatory activity or mortality. Survived rats were euthanized in a CO2 chamber, and a general macroscopic observation of tissues was done. If rats died at 2000 mg/kg, the dosage was reduced to 1000 or 100 mg/kg. The parameter of LD50 was calculated according to the modified practical method reported by Lorke (1983). In this assay, a minimum number of experimental animals is used to calculate acute toxicity by applying a geometric mean over the doses for which 0% and 100% mortality was found.

Statistical analysis

Data were expressed as the mean ± standard error of the mean (S.E.M.) of 6 rats as indicated in the figure legends. Area under the curve (AUC) was calculated from the temporal course curves to describe the dose-anti-inflammatory response of the AEBS, SFN, INDO, or KET compared to the vehicle group. Data were analyzed using one or two-way analysis of variance (ANOVA) as appropriate followed by Dunnett’s post hoc test to compare treatments versus control group (vehicle by i.p. or p.o., respectively). A value of p < 0.05 was considered significant.