Animals

Adult wild-type zebrafish (Danio rerio) of both sexes were obtained from a local supplier and acclimated for 4 weeks before the experiments were conducted at the bioterium at Universidade Comunitária da Região de Chapecó, Brazil. Zebrafish embryos were obtained from natural mating of adult zebrafish maintained in bred tanks. Fertilized eggs were collected, washed with system water (reverse osmosis water equilibrated with Instant Ocean salts), freed of debris, and transferred to sterile cell culture plates. Plates were maintained in an incubator at 28.5 °C and monitored daily until 7 dpf. Adult animals were housed in thermostated thanks filled with unchlorinated water constantly aerated at a targeted temperature of 26 ± 2 °C. Fish were kept under a 14–10 h light/dark cycle photoperiod and fed daily, three times a day, with fish food that was supplemented with live artemia. Euthanasia of animals was performed after the tests and was practiced by immersion in a tricaine solution. The protocol was approved by the Ethics Committee for Animal Use (CEUA) of Unochapecó (Protocol #014/2016) and followed the guidelines of Conselho Nacional de Controle de Experimentacão Animal (CONCEA).

Materials

Curcumin, pentylenetetrazole (PTZ), tricaine and valproate (VPA) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Curcumin Micronization with the Solution Enhanced Dispersion by Supercritical Fluids Technique

The Solution Enhanced Dispersion by Supercritical Fluids technique (SEDS), experimental equipment and procedure used to micronize curcumin with supercritical CO 2 as anti-solvent, is described in detail by previous studies25,26. The process parameters adopted in the present report were based on optimized trans-resveratrol and N-acetylcysteine micronization data provided by Aguiar et al.: solute concentration of 20 mg∙mL−1, temperature at 35 °C, anti-solvent flow rate of 20 mL∙min−1, solution flow rate of 1 mL∙min−1 and operating pressure of 8 bar15,27.

Particle Characterization

In order to characterize the particles, the following analyzes were performed: Morphology by Scanning Electron Microscopy (SEM) and determination of particle size by the software Meter Size (version 1.1)15.

Differential scanning calorimetry

The melting point of the compound was determined using a system of differential scanning calorimetry (Jade-DSC, Perkin Elmer). The samples (5–10 mg) were prepared in an aluminum pan, and DSC measurements were performed by heating at 30 to 200 °C at a rate of 10 °C/min in an inert atmosphere (N2 flow: 10 mL·min−1)15.

Treatments

All animals received the respective treatment 30 min before the PTZ exposure. In order to verify the zebrafish response to a classic AED in our PTZ-induced seizure model, we treated a group of animals with VPA. All groups were treated and analyzed in an identical manner. Curcumin and VPA concentrations and doses were selected based on preliminary studies conducted in our laboratory.

Zebrafish larvae (n = 20) were individually placed in 6 well plates (1 larva per well with 5000 μL of solution) and submitted to treatment with 0.1% DMSO (control group), curcumin at 1 μM, micronized curcumin at 1 μM or VPA at 3 mM for 30 min. In adult animals, the 1% DMSO (control group), curcumin at 0.50 mg/kg, micronized curcumin at 0.50 mg/kg and VPA at 100 mg/kg were applied by intraperitoneal (i.p.) injection. I.p. injections were given using a 3/10-ml U-100 BD Ultra-FineTM Short Insulin Syringe 8 mm (5/16) × 31 G Short Needle (Becton Dickin-son and Company, New Jersey, USA) according to the protocol established by Phelps et al.28. Anesthesia of adult animals prior to the injection was given by their immersion in a 10% tricaine solution until the animals showed lack of motor coordination and reduced respiration rate. After the injection, the animals were placed in a separate tank with aerated unchlorinated tap water (26 ± 2 °C) to recovery from anesthesia.

Locomotor and exploratory activity

In order to analyze the effects of curcumin and micronized curcumin on locomotor and exploratory activity, all animals were individually submitted to the novel tank test29,30. Animal behavior was recorded during the 30 min following curcumin and micronized curcumin and prior to PTZ exposure. The same protocol was applied to verify the effects of VPA. Larvae were individually placed in 6 well plates (1 per well, with 5000 μL of solution). Adult animals were individually placed in glass tanks (12 cm × 8 cm × 13.5 cm, length × width × height). The animal behavior was registered by a video camera for 30 min and further analyzed using the ANY-Maze® recording software (Stoelting Co., Wood Dale, IL, USA) to track the locomotor and exploratory behavior of the animals. To analyze the behavior, the larvae novel tank apparatus was divided into center and periphery. The adult novel tank apparatus was divided into bottom, middle and upper. Distance traveled and zone line crossing were analyzed.

PTZ-induced seizures

To induce seizures, zebrafish were individually exposed to 3 mM PTZ19. Larvae were individually exposed to PTZ in 6-well plates. PTZ solution was applied in the larval medium in order to obtain a final concentration of 3 mM in a total volume of 5000 μl. Adult zebrafish were individually exposed to 3 mM PTZ by their immersion in 250 mL beakers. All PTZ treatments were videotaped and evaluated later by trained observers. The seizure-like behavior was classified according to each stage: stage I—dramatically increased swimming activity, stage II—whirlpool swimming behavior, and stage III—clonus-like seizures followed by loss of posture, when the animal falls to one side and remains immobile for 1–3 s, as previously reported for zebrafish19. The animals were submitted to the PTZ treatment until they reached stage III, which corresponds to the tonic–clonic seizure stage in zebrafish, or until 600 s.

The occurrence of each seizure stage and the latency to the first behavioral sign of each seizure stage were analyzed. Experiments were performed in triplicate, on different days. Experimental groups consisted of 20 zebrafish larvae and 18 adult zebrafish, in an equal number of males and females. All tests were done in the morning.

Statistical analysis

Normality of the data distribution was confirmed with the Kolmogorov-Smirnov test. Obtained data about the occurrence of each seizure stage was analyzed using Kruskal-Wallis followed by Dunn’s test, and results are expressed as a median with interquatile range. Considering the latency to reach each seizure stage and behavioral parameters, data was analyzed using One-way (ANOVA) followed by Dunnett’s post-test. These results are expressed as a mean ± standard error of the mean (S.E.M.).