Xylazine hydrochloride

The following drugs, salts, and solutions were used: ketamine hydrochloride (Syntec, São Paulo, SP, Brazil), xylazine hydrochloride (Syntec, São Paulo, SP, Brazil), isoflurane (BioChimico, Rio de Janeiro, RJ, Brazil), and heparin (Hipolabor, Belo Horizonte, MG, Brazil). Phenylephrine, sodium nitroprusside (SNP), acetylcholine (ACh), NaCl, KCl, CaCl₂, MgSO₄, NaHCO₃, KH₂PO₄, dextrose, ethylenediaminetetraacetic acid, cholesterol, and colic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the other reagents were obtained in analytical grade.

The following drugs and salts were used: ketamine hydrochloride, xylazine hydrochloride (Syntec, São Paulo, SP, Brazil), heparin (Hipolabor Pharmaceuticals, Belo Horizonte, MG, Brazil), phenylephrine (Phe), sodium nitroprusside (SNP), hydrochlorothiazide (HCTZ), angiotensin I, angiotensin II, endothelin 1, acetylcholine chloride (ACh), indomethacin, NaCl, KCl, CaCl₂, MgSO₄, NaHCO₃, KH₂PO₄, dextrose, and ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, Missouri, USA). All other reagents were obtained with analytical grade.

The following materials were used: acetic acid and absolute ethyl alcohol (Neon, São Paulo, Brazil), L-glutamic acid (glutamate) (Vetec, Rio de Janeiro, Brazil), capsaicin, cinnamaldehyde, indomethacin (Sigma-Aldrich, St. Louis, MO, USA), ketamine hydrochloride, xylazine hydrochloride (Syntec, São Paulo, Brazil), naloxone hydrochloride (Cristália, São Paulo, Brazil), formalin (Dinâmica, São Paulo, Brazil), sodium hydroxide (Nuclear, São Paulo, Brazil), menthol (A Essência, Santa Catarina, Brazil), and tween 80 (Labsynth, São Paulo, Brazil). The drugs were dissolved in 0.9% NaCl solution (saline) before administration, except cinnamaldehyde (1% tween 80 in saline), glutamate (saline solution at pH 7), and menthol (1.6% absolute ethyl alcohol + 0.01% tween 80 in saline). capsaicin was prepared from a 0.5% capsaicin stock solution dissolved in absolute ethyl alcohol, and the 0.1% solution was prepared at the time of use by mixing the stock solution with tween 80 and 2:1:7 saline solution.

Female BALB/c mice (4–6 weeks-old) ranging from 20 to 25 g were randomly distributed into five groups. Animals of the groups 1 to 3 were subcutaneously (SC) infected with 1 × 10⁴ promastigote forms of L. amazonensis in the left hind footpad, while animals of the groups 4 and 5 were kept uninfected. As soon as the lesions appeared, on the 28^th day after the inoculum, all animals were weighed and the thickness of their footpads, right and left, was measured with a caliper (0.1 mm, Schnelltäster, HC Kroplin). Then, intralesional treatment (IL) with amentoflavone (0.5 mg/kg/dose) was initiated, through subcutaneous injection in the lesion site (groups 1 and 4). N-metil glucamine (64 mg Sb⁵⁺/kg/dose) was used as a positive control, and amentoflavone vehicle (10% Ethanol/10% Cremophor/1% DMSO/PBS) as a negative control of treatments (groups 3 and 5, respectively). All treatments were performed in five doses, administered 4 days apart. The animals’ weight and footpad thickness were monitored weekly. One week after the last treatment dose, animals were weighed and the lesion was measured just before being euthanized with Xylazine Hydrochloride (30 mg/kg, Syntec) associated with Ketamine Hydrochloride (300 mg/kg, Syntec). After euthanasia, tissue and blood samples were collected for subsequent analysis.

Mice were weighted, submitted to anesthesia with 10% ketamine hydrochloride associated to 2% xylazine hydrochloride (Syntec, Santana de Parnaíba, Brazil). Blood samples collection was performed by cardiac puncture. After animal euthanasia, spleen, lungs, and liver were removed at days described in experimental hyalohyphomycosis item. The lung and liver specimens obtained from mice were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and Grocott’s (methenamine silver) and the photographs were taken by Leica DM 1000 (Leica, Wetzlar, Germany).

The following materials were obtained from commercial sources: castor oil, prostaglandin E2, charcoal, atropine, carbachol (CCh), Splittgerber’s reagent, cholera toxin, monosialoganglioside-GM1, and 3,3’,5’,50-Tetramethylbenzidine, from Sigma-Aldrich, Inc. (St Louis, MO, USA); bethanechol chloride, loperamide hydrochloride, and naloxone hydrochloride from Janssen-Cilag Pharmaceutics LTDA, Brazil and CRISTÁLIA Pharmaceutical Chemicals products LTDA, Brazil; Xylazine hydrochloride and ketamine hydrochloride, were obtained from Syntec (Cotia, SP, Brazil). All other chemicals used were of analytical grade and obtained from standard commercial suppliers. All drugs were dissolved in saline or phosphate-buffered saline (PBS).

This study used female Swiss mice aged 6 to 8 weeks weighing 17–23 g. They were acquired from the Central Animal Facility of the Federal University of Goiás and placed in the Laboratory of Biomolecules and Vaccines (LaBVac) pre-clinical testing room. The mice were housed in a pathogen-free primary enclosure with free access to food and water; the enclosure was maintained at 24 °C, with odor and light control with a light and dark cycle every 12 h. All animals in this experiment were previously dewormed with access to a 1:20 Ivermectin^® solution for seven days. The experiments were conducted following ethical recommendations established by the law of procedures for the scientific use of animals, being approved for execution by the Animal Ethics and Research Committee of UFT under protocol number 23101.002359/2020-31. Every effort was made to avoid suffering or undue pain; the mice were monitored for weight loss and gain and clinical manifestations such as lethargy, hypothermia, and difficulty breathing. The animals were euthanized using ketamine (300 mg/kg) (Vetbrands^®, Rio de Janeiro, Brazil) and xylazine hydrochloride (22.5 mg/kg) (Syntec^®, Piracicaba, Brazil) [17 (link),18 (link),19 (link)].