To assess the PARP-1 or PARP-2 inhibitory activity of FR247304, 3-AB, and PJ34, PARP activity is evaluated with minor modifications. PARP enzyme assay is carried out in a final volume of 100 μL consisting of 50 mM Tris-HCl (pH 8.0), 25 mM MgCl₂, 1 mM dithiothreitol, 10 μg activated salmon sperm DNA, 0.1 μCi of [adenylate-³²P]NAD, 0.2 units of recombinant human PARP for PARP-1 assay or 0.1 units of recombinant mouse PARP-2 for PARP-2 assay, and various concentrations of FR261529 or 3-AB. The reaction mixture is incubated at room temperature (23°C) for 15 min, and the reaction is terminated by adding 200 μL of ice-cold 20% trichloroacetic acid (TCA) and incubated at 4°C for 10 min. The precipitate is transferred onto GF/B filter and washed three times with 10% TCA solution and 70% ethanol. After the filter is dried, the radioactivity is determined by liquid scintillation counting.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

PC12 cell cultured are grown in Dulbecco's modified Eagle's medium supplemented with 5% (v/v) fetal calf serum, 5% (v/v) horse serum, and a 1% (v/v) penicillin-streptomycin antibiotics mixture. Cells are grown in an atmosphere of 95% air and 5% CO₂ at 37°C. For all experiment, cells are seeded at a density of 4×10⁴ cells/well in 96-well culture plates and allowed to attach overnight. For assessment of cell viability, hydrogen peroxide-induced cytotoxicity is quantified by a standard measurement of LDH release with the use of the LDH assay kit. Briefly, 6 h after hydrogen peroxide exposure, 20 μL of medium of each well is collected, and the solution prepared from LDH assay kit is added. After incubation at room temperature for 30 min, the reaction is stopped by addition of 1 N HCl, and absorbance is measured at 450 nm using a microplate reader.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[1]
For transient focal ischemia, 9- to 10-week-old male Wistar rats (weighing 274-380 g) are used. FR247304, PJ34, or 3-AB, which is suspended with 0.5% methylcellulose, is administered at doses of 10 and 32 mg/kg for FR247304, 3.2 and 10 mg/kg for PJ34, or 32 and 100 mg/kg for 3-AB intraperitonially twice at 10 min before MCA occlusion and 10 min before recirculation. The administration volume is adjusted to 2 mL/kg.
Mice^[2]
Male Swiss albino mice (27-32 g) are used. The PARP inhibitor, PJ34 (1.25, 12.5 or 25 mg/kg) is dissolved in isotonic saline (NaCl, 0.9%) and injected intraperitoneally, in a volume of 10 mL/kg, 15 min before ischemia and again 4 h after the onset of ischemia. Control ischemic mice and sham animals are given vehicle (saline). Naive animals are also included in the studies.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Iwashita A, et al. A novel and potent poly(ADP-ribose) polymerase-1 inhibitor, FR247304 (5-chloro-2-[3-(4-phenyl-3,6-dihydro-1(2H)-pyridinyl)propyl]-4(3H)-quinazolinone), attenuates neuronal damage in in vitro and in vivo models of cerebral ischemia. J Ph  [Content Brief]

  [2]. Haddad M, et al. Anti-inflammatory effects of PJ34, a poly(ADP-ribose) polymerase inhibitor, in transient focal cerebral ischemia in mice. Br J Pharmacol. 2006 Sep;149(1):23-30.  [Content Brief]