2 3 5 triphenyltetrazolium

Two types of the bacterial symbiont, Photorhabdus, were isolated from the two entomopathogenic nematodes, Heterorhabditis indica (HRM1), isolated from Alexandria, Northern Egypt, and Heterorhabditis sp. (HS1), isolated from Ras-Sidr in South Sinai of Eastern Egypt, as detailed in (El-Sadawy et al. 2016 ). Briefly, bacteria were isolated from their symbiotic nematodes according to Woodring and Kaya (1988) . For each subculture, the phase status was determined by culturing on NBTA agar [2.3% nutrient agar (Difco), 0.0025% bromothymol blue (Merck), 0.004% 2,3,5-triphenyltetrazolium (Merck)]. Phase I colonies are blue on NBTA while phase II colonies are red. Twenty infective nematode juveniles were surface sterilized for 10min in 1.0% sodium hypochlorite, washed in sterile distilled water, transferred to a Petri dish containing 5ml of TSBYE [3% tryptic Soy broth (Difco), 0.5% yeast extract (Difco)], and grinded using grinder pistol. The plates were incubated at 30 °C for 24h and streaked on NBTA plates [2.3% nutrient agar (Difco), 0.0025% bromothymol blue (Merck), 0.004% 2,3,5-triphenyltetrazolium (Merck)]. The presence of Photorhabdus colonies was confirmed by dye adsorption on NBTA plates, production of luminescence, and antibiotic activity. The isolated bacteria were then maintained on NBTA plates at 10 °C and subcultured weekly.

Adult male Sprague-Dawley rats weighing 250–300 g were purchased from Chongqing Medical University. Rats were kept under controlled conditions (25 ± 1°C, 60–65% humidity, and 12/12 h light/dark cycle) for the duration of the study. Water and food were available ad libitum. All experiments were carried out in accordance with the National Institute of Health guideline requirements in China.
Cur, Mithramycin (MTM), and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma (St. Louis, USA). Cur (300 mg/kg) was dissolved in normal saline with 1% dimethylsulfoxide (DMSO) and injected intraperitoneal (IP) 1 hour after middle cerebral artery occlusion following established protocols [16 (link)]. MTM (250 μg/kg) was administered at the start of reperfusion [17 (link)]. Small interfering RNA (siRNA) targeted to Prdx6 (Prdx6-siRNA) and a scrambled RNA (scrRNA) controls were purchased from GenePharma Co., Ltd. (Shanghai, China). The sequence and antisequence were 5′-CUUCCACGAUUUCCUAGGATT-3′ and 5′-UCCUAGGAAAUCGUGGAAGTT-3′, respectively. 24 hours before middle cerebral artery occlusion (MCAO) surgery, 5 μl of either Prdx6-siRNA or scrRNA was applied by intracerebroventricular injection at a rate of 0.2 μl/min. The stereotactic coordinates were 1.5 mm lateral and 1.1 mm posterior to the bregma and 4.5 mm below the surface of the skull.