Transient Middle cerebral artery occlusion (tMCAO) and AGmex administration

The most used tMCAO model is known as Koizumi’s model (Koizumi et al. 1986). In this study, we adapted Koizumi’s method to established tMCAO models, with some modifications (Lee SE et al. 2018).

Mice were allocated (at least nine mice per group) to a sham-operated group; a tMCAO-operated, but not AGmex-treated group (the MCAO group); and tMCAO-operated and AGmex-treated groups, which were pre-treated with AGmex at 300, 1000, or 3000 mg/kg at 1 h (single administration) before MCAO or at 1 h and 24 h (two administrations) before MCAO, or with 1000 mg/kg of AGmex at 1 h after MCAO, respectively. Mice in the AGmex-treated groups were administered 0.5 mL of each concentration daily. Mice in the sham-operated and MCAO groups received the same amount of physiological saline as that administered with AGmex in the AGmex-treated groups. Each group consisted of at least three mice per group. Nimodipine (ND; the positive control) was also administered as a single dose of 60 mg/kg bw at 1 h post-tMCAO, as previously reported (Li JH et al. 2013; Zhao et al. 2018). A schematic of the experiment is shown in Figure 2.

Experimental schedule of the 2 h transient middle cerebral artery occlusion (tMCAO) model. The mice were acclimated for 1 week in our animal facility before study commencement. Pre-treatment (Pre-Tx.) and post-treatment (Post-Tx.) studies were carried out to determine the timing of AGmex administration. TTC; 2,3,5-triphenyl-tetrazolium chloride; EB; Evans blue.

In this study, researchers who performed surgery on experimental mice, researchers who conducted several assays, and a researcher who performed statistical analyses, were separately assigned. The researchers who participated in each step, then, analysed the final study results together.