Neomycin trisulfate salt hydrate

Lysine fixable 3 kDa and 10kDa-FITC or Texas Red-labeled dextrans were purchased form ThermoFisher scientific Inc (Waltham, MA.). Dextrans were diluted in Hanks' balanced salt solution without Ca²⁺ and Mg²⁺ (HBSS-CFM) at a final concentration of 10 mg/mL, aliquoted in black opaque tubes and stored at −30°C until use. As indicated by the manufacturer, the actual molecular weights of dextran molecules in each preparation has a distribution, and contains polymers with molecular weights ranging from 1.5 to 3.0 kDa (including the dye) in the case of 3 kDa dextran-TR, and 50% of the dextran molecules contains a dye, on average.
Hank’s balanced salt solution (HBSS), HBSS-CFM, phosphate buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), DMEM media without calcium, and Leibovitz’s L-15 medium with GlutaMAX were also purchased from ThermoFisher scientific Inc. Cell impermeant BAPTA tetrapotassium salt (Molecular Probes, ThermoFisher scientific, Inc.) was dissolved at 50 mM in water. A stock solution of 30 mM benzamil hydrochloride hydrate (Sigma-Aldrich, St. Louis, MO) was prepared in DMSO. Amiloride hydrochloride (EMD MILLIPORE, Burlington, MA) was dissolved at 150 mM in DMSO. Aminoglycosides, dihydrostreptomycin sesquisulfate, and neomycin trisulfate salt hydrate (Sigma-Aldrich) were dissolved at 50 mM in water.

The rearing diet contained a mixture of powdered oat flakes, wheat germ and Pangamin, a dried yeast biomass (Rapeto, Bezdružice, CZ) (10/10/1 w/w/w). The rearing diet (control) was powdered by blender, sieved at 0.5 mm sieves and sterilized by heating to 70°C for 0.5 h. The diet of 50±5 mg was placed to every IWAKI chamber. The antibiotics, ampicillin sodium salt (cat No. A0166, Sigma-Aldrich, Saint Louis, USA), neomycin trisulfate salt hydrate (cat No. N6386, Sigma-Aldrich) and streptomycin sulfate salt (cat No. S9137, Sigma-Aldrich) were added to the rearing diets, separately. The treatments included antibiotic concentrations of 0.1, 1, 10, and 30 mg of antibiotics per g of diet (mgg⁻¹). The control was the rearing diet without supplementation.

Male six-week-old Fischer 344 rats (CLEA Japan, Inc., Osaka, Japan) were housed in a room under controlled temperature and a 12/12 h light-dark cycle. The animals were divided into the following three experimental groups and fed for 8 weeks: i) Choline-deficient amino acid diet (CDAA; n=10); ii) choline-deficient amino acid diet plus antibiotics (CDAA+AB; n=10) and iii) choline-supplemented amino acid diet (CSAA; n=5). All rats were sacrificed at the end of week 8. For selective intestinal decontamination, poorly absorbable antibiotics (1 g/l polymyxin B sulfate salt (Fluka, Buchs, Switzerland) and 3 g/l neomycin trisulfate salt hydrate (Sigma-Aldrich, St. Louis, MO, USA) were administered to the rats in the CDAA+AB group by adding them to drinking water during the experimental period, excluding the first and fifth week. All animal procedures were performed according to standard protocols and in accordance with the standard recommendations for the proper care and use of laboratory animals. This study was approved by the ethics committee of Nara Medical University, Kashihara, Japan.