Ketamin

The following reference chemicals were used in the experiments: butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), resazurin, 2,3,5-triphenyltetrazolium chloride (TTC), chloramphenicol, nystatin, quercetin, pyrogallol Cremophor EL, all provided by Sigma-Aldrich (St. Louis, MO, USA); ascorbic acid (AAc), purchased from Dinâmica (Diadema, São Paulo, Brazil); linoleic acid, obtained from Vetec (Duque de Caxias, Rio de Janeiro, Brazil); 2,2-diphenyl-1-picrylhydrazyl (DPPH), provided by FLUKA (St. Louis, MO, USA); silver sulphadiazine 2% (União Química, Brasília, Distrito Federal, Brasil); paracetamol (Johnson & Johnson, São José dos Campos, São Paulo, Brazil); dipyrone (Sanofi Aventis, São Paulo, Brazil); ketamin (Cristália, Itapira, São Paulo, Brazil); and xylazine (Intervet Schering-Plough, São Paulo, Brazil). Stock solutions of these chemicals were prepared with suitable solvent and dilutions were made fresh on the day of experiment. Mueller-Hinton Agar and Mueller-Hinton Broth culture mediums were both purchased of HiMedia (Mumbai, India) and prepared with sterile distilled water.

In order to evaluate the ability of hiPSC to develop teratomas, approximately 3x10⁶ hiPSCs from ALS and non-ALS patients were injected subcutaneously into the dorsal flanks of nude rats anesthetized with ketamin (Cristália, Brazil)-xilazin (Vetbrands, Brazil; 62.5 mg/kg). Teratomas were allowed to expand for about 6 weeks. They were visualized and dissected by means of surgical procedure, fixed overnight in 4% paraformaldehyde and transferred to 70% ethanol until they were embedded in paraffin. Sections were stained with hematoxylin and eosin for microscopy analyses. Protocols for animal use were previously approved by the School of Medicine of the University of São Paulo Institutional Animal Care and Use Committee.

The animals were submitted to anesthetic procedure with 2% xylazine hydrochloride
(Xilazin®, Syntec, Santana de Parnaíba, SP, Brazil) (10 mg.kg^-1)
associated with 5% dextrocetamine hydrochloride (Ketamin®, Cristália, Itapira,
SP, Brazil) (25 mg.kg^-1), prepared by combining 0.5 mL of xylazine
(10 mg) with 0.5 mL of Ketamine (25 mg) to a volume of 1 mL administered by
intraperitoneally (1 mL.kg^-1)8 (link).
After trichotomy of the dorsal region and skin asepsis, each animal was submitted
to two circular excisions, with 2 cm in diameter, 2 cm distant from each other,
in median dorsal plane, limited in depth by muscle aponeurosis, performed with a
scalpel (handle and blade number 15)8 (link).
The experiment was randomized into naïve (health skin), untreated (NT) or
DPG-treated (DPG) animals and divided into groups following site (proximal or
distal), treatment (untreated or DPG-treated) and day of euthanasia (14 or 21).
After excision, the proximal wound was untreated, and distal wound was daily
treated with 0.1 mL of 2% DPG cream, for seven days, starting 24 hours after
surgery. Both proximal (untreated) and distal wound (DPG-treated) did not
require bandages and healed by second intention, based on literature8 (link), which used tretinoin for the same
purpose. All animals were euthanized after the wound scar excisions at day 14 or
21, as described in Fig. 1.