Aminoguanidine hydrochloride ag

The formation of AGEs in BSA was induced by glucose according to a previously published method with slight modifications [34 (link)]. Briefly, 10 mg/mL BSA (PanReac AppliChem, Darmstadt, Germany) and 0.5 M d-(+)-glucose (Sigma-Aldrich, Saint Louis, MO, USA) were prepared in 0.1 M phosphate buffer (pH 7.4). CFE (100 to 500 µg/mL) and 4.5 mM aminoguanidine hydrochloride (AG, Sigma-Aldrich, Saint Louis, MO, USA) were incubated with BSA and glucose at 37 °C for 28 days. AG was used as a positive control for inhibiting glycation. Incubated samples were used to detect the formation of AGEs, CML, and carbonylated protein at 14, 21, and 28 days after incubation. Fluorescent AGEs in glucose-modified BSA were detected with an excitation wavelength of 340 nm and an emission wavelength of 430 nm. The percentage of glycation was calculated as the fluorescence intensity of the sample compared to the control (BSA +/Glucose +).

Aminoguanidine hydrochloride (AG; Sigma-Aldrich, St. Louis, USA), the inhibitor of iNOS, was intraperitoneally administered to mice (60 mg/kg in 100 μl of sterile saline solution) daily starting on the day preceding the infection and finishing a day before the desired time points (see below). The dose causes 85% inhibition of the lipopolysaccharide-induced increase in total plasma nitrite/nitrate concentration and is safe for mice [48 (link)]. Control mice received the sterile saline solution only and were infected in the same manner. At the desired time points (18 hpi for pinnae samples, 3 and 7 dpi for CNS samples), both control and AG-treated mice were anaesthetized by isoflurane and bled out. Individual pinnae or spinal cords and cerebella were extracted and separately torn apart by sharp forceps in the PBS. Schistosomula were then collected under a stereomicroscope, counted, fixed with hot water and measured (Quick Photo Micro 3.0).
Additionally, the effect of AG treatment on the nervous tissue myelination was examined 0 and 7 dpi. Spinal cords of control and AG treated mice were processed for immunohistochemistry following the aforementioned protocol. Rabbit anti-mouse MBP polyclonal antibody (1:1000, Thermo Fisher Scientific) was used and sucrose antigen retrieval was not performed for this staining.

Glucose, fructose, and 2,4-dinitrophenyl hydrazine (DNPH) were purchased from Ajax Finechem (Taren Point, Australia). Catechin, gallic acid, sodium azide, Nitroblue tetrazolium (NBT), aminoguanidine hydrochloride (AG), guanidine hydrochloride, Thioflavin T (ThT), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and l-cysteine were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA) was purchased from Merck (Darmstadt, Germany). OxiSelectTM CML ELISA kit was purchased from Cell Biolabs (San Diego, CA, USA). All other reagents used were of analytical grade.