Ketamine 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.

For the experiments, the following were used: ketamine hydrochloride and xylazine (from Syntec, São Paulo, SP, Brazil), 4-aminopyridine (4-AP), acetylcholine chloride (ACh), atropine, CaCl₂, dextrose, ethylenediaminetetraacetic acid, glibenclamide, HOE-140, KCl, KH₂PO₄, NaCl, NaHCO₃, MgSO₄, Nω-Nitro-L-arginine methyl ester (L-NAME), indomethacin, phenylephrine (Phe), sodium deoxycholate, and tetraethylammonium (TEA) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and heparin from Hipolabor (São Paulo, SP, Brazil)

The following drugs, salts, and solutions were used: xylazine and ketamine hydrochloride (from Syntec, São Paulo, SP, Brazil), heparin (from Hipolabor, Belo Horizonte, MG, Brazil), and acetylcholine chloride, phenylephrine, indomethacin, Nω-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside, NaCl, KCl, NaHCO₃, MgSO₄, CaCl₂, KH₂PO₄, dextrose, and ethylenediaminetetraacetic acid (EDTA) (all purchased from Sigma-Aldrich, Saint Louis, MO, USA).

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.

Xylazine, ketamine hydrochloride (Syntec, São Paulo, SP, Brazil), and heparin (Hipolabor, Belo Horizonte, MG, Brazil) were used in the experiments. The reagents acetylcholine chloride, angiotensin II, indomethacin, metoprolol, NaCl, MgSO₄, CaCl₂, dextrose, phenylephrine, KH₂PO₄, ethylenediaminetetraacetic acid, 2′,7′-dichlorofluorescein-diacetate, 5,5′-dithiobis, sodium nitroprusside, bovine serum albumin, KCl, NaHCO₃, reduced glutathione, Tris-HCl, xylenol orange, and ethylenediaminetetraacetic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). For LC-DAD-MS analyses, methanol, acetonitrile, and formic acid were HPLC grade and purchased from J.T. Baker (Phillipsburg, NJ, USA).

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.