Ischemic stroke model (MCAO)

The ischemic stroke model (focal ischemia) was performed on seven weeks old male mice by transient (60 min) right middle cerebral artery occlusion (MCAO) and reperfusion, using a 6-0 coated filament (Doccol, Redlands, CA, USA) as previously described³⁶. The regional cerebral blood flow was monitored by laser-Doppler flowmetry (Perimed, Craponne, France) to control MCAO severity and reperfusion. Animals presenting with sustained CBF reduction >70% during ischemia or a severe brain hemorrhage after MCAO were excluded from the study (<1%). Mice received MLC901 in post-treatment until the sacrifice of animals. Sham-operation was performed by inserting the thread into the common carotid artery without advancing it to occlude MCA. The animals were allowed to regain full consciousness on a nursing cage during 24 hours before returning to the home cage. The researchers who did the MCAO surgery were blinded to the treatment code.