S-MTC

Cell experiment:

Mixed cortical glial and neuronal cultured cells are prepared from E15 to E18 embryos obtained from Spargue-Dawley rats. On day 7 after plating, the culture medium is removed and replaced with freshly prepared culture medium in the presence of either Aβ1-42 (1, 5, 10, or 20 μM), Aβ42-1, or peroxynitrite (100 or 200 μM) with or without either NG-nitro-L-arginine (L-NOARG,10 or 100 μM), S-MTC (10 or 100 μM), N-iminoethyl-L-lysine (10 or 100 μM), N-(3-(aminomethyl)benzyl)acetamidine (1400W,1 or 5 μM), 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO,10 or 100 μM), or 6-hydorxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (10 or 100 μM) alone or in combination. The cultured cells are then incubated for 20 h. For the time-course studies, the cultured cells are pre-treated with the described culture medium containing Aβ1-42 (10 μM). Either L-NIL (100 μM), L-NOARG (100 μM), 1400W (5 μM), S-MTC (100 μM), carboxy-PTIO (100 μM) or Trolox (100 μM) are administered at 1, 4, and 8 h later. Assessments are carried out 20 h after Aβ1-42 administration. The viability of cultured cells is evaluated by using MTT and neutral red colorimetric assays. MTT reduction and NR uptake are quantified at 570 and 540 nm, respectively, by using a micro-plate reader[1].

Animal experiment:

mice[2]Male NIH Swiss mice, weighing 18-22 g, are used. S-MTC (1.0 μg/mouse) is administered i.c.v. (15-min pretreatment time). In one set of experiments (#1, #2, and #3), opioid antagonists and NOS-inhibitors are administered 15–30 min prior to the 60-min HBO2 treatment (180 min prior to antinociceptive testing). In another experiment (#4), opioid antagonist and NOS-inhibitor pretreatment is administered 60 min following cessation of the 60-min HBO2 treatment (15–30 min prior to antinociceptive testing). For i.p. or s.c. pretreatments, the volume of injection is 0.1 mL/10 g body weight with control animals receiving an i.p. or s.c. injection of vehicle (sterile saline) only. For i.c.v. pretreatments, the volume of microinjection is 5.0 μL per mouse with control animals receiving an i.c.v. microinjection of vehicle (sterile saline) only.Rats[3]Male, Sprague-Dawley rats (350-450 g) are used. On the day after catheterisation (day 1), animals (n=7) receive bolus i.v. injections (0.1 mL) of either saline (vehicle), and 0.3 and 3 mg/kg S-MTC (n=4), or 0.1, 1 and 10 mg/kg S-MTC (n=3). On day 3, the dose regimen is switched to ensure that each animal has received all the doses of S-MTC. On each day, drugs are given in ascending dose-order, and at least 60 min is allowed between doses. The intervening day (day 2) is allowed for wash-out of any drug effects.

References:

[1]. Law A, et al. Neuroprotective and neurorescuing effects of isoform-specific nitric oxide synthase inhibitors, nitric oxide scavenger, and antioxidant against beta-amyloid toxicity. Br J Pharmacol. 2001 Aug;133(7):1114-24.
[2]. Zelinski LM, et al. A prolonged nitric oxide-dependent, opioid-mediated antinociceptive effect of hyperbaric oxygenin mice. J Pain. 2009 Feb;10(2):167-72.
[3]. Wakefield ID, et al. Comparative regional haemodynamic effects of the nitric oxide synthase inhibitors, S-methyl-L-thiocitrulline and L-NAME, in conscious rats. Br J Pharmacol. 2003 Jul;139(6):1235-43.