2.3 Experimental design
The present experiment was divided into two parts (Fig. 1B). In part 1, effects of FFA on CA/CPR-treated WT mice were investigated. At 1 h after ROSC, mice were randomized to receive FFA or vehicle by using random number table (Fig. 1A). Mice in the FFA group were given FFA (12.5 mg/kg in sterile saline, 4% PEG-400, 2% DMSO; Sigma, St. Louis, MO) via intraperitoneal injection once daily for 1 week [24], while mice in the vehicle group received equivalent volume of vehicle solution. Forty mice with successful resuscitation were followed up for 7 days to evaluate survival, neurological function, and histological injuries. The other 45 mice were used to describe the neuroprotective effects by detecting cellular apoptosis, neuronal degeneration, BBB disruption, edema formation and neuroinflammation markers at day 3 after CA/CPR. In part 2, we explored whether gene deletion of Trpm4 improved neurologic outcome after CA/CPR and whether FFA interfered with the TRPM4 channel. Twenty-six Trpm4−/− mice that underwent CA/CPR were randomly allocated to receive vehicle or FFA, and 13 CA/CPR-treated WT mice given vehicle were set as the control group (Fig. 1A). In this part, expression of TRPM4 was evaluated at 24 h after CA and other experiments were in line with that of part 1.