Experimental procedures

Rats were anesthetized with an intraperitoneal injection of 10 % chloral hydrate under spontaneous inspiration, and the body temperature was continuously monitored at 37 ± 0.5 °C during the surgical procedures. For all rats undergoing MCAO, the left middle cerebral artery (MCA) was occluded for 60 min. In detail, rats were immobilized by a tooth holder and with binding of all limbs, followed by the insertion of a 4.0 silicon-coated polypropylene suture into the left internal carotid artery (ICA) through the external carotid artery (ECA) and common carotid artery (CCA) to block blood flow to the MCA. After 60 min, the filament was withdrawn from the ICA to allow reperfusion. Two control subgroups experienced identical operations but without MCA occlusion. MRI data were acquired under anesthetized circumstances to examine brain edema and intracerebral bleeding of the left brain hemisphere 24 h later for the control and day 1 MCAO subgroups, 3 days later for the day 3 MCAO subgroup, 7 days later for the day 7 MCAO subgroup, and 14 days later for the day 14 MCAO subgroup. Additionally, a functional examination was performed repetitively at the same time points prior to MRI. Immediately after MRI scanning, group 1 received 50 mM of a MnCl2 solution (267.9 μmol/kg) delivered via syringe at a rate of 1.2 ml/h through the tail vein. Group 2 received 0.2 μl of 1 M MnCl2, injected with a 2.0-μl Hamilton syringe at a rate of 0.05 μl/min via the burr hole. MRI data were acquired again 4 days later for group 1 and 2 days later for group 2. Finally, rats of group 1 were sacrificed for immunofluorescence staining by decapitation under deep anesthesia.