Middle cerebral arterial occlusion model and experiment groups

Twenty male Sprague Dawley rats (250–320 g; Charles River Laboratories, USA) were subjected to transient (60 min) middle cerebral arterial occlusion (MCAO) via intraluminal vascular occlusion, as previously described [19, 20]. Rats were anesthetized initially with 3.5% isoflurane mixed with room air and maintained at 2.0% isoflurane during surgery, and 1.5% during MRI. The left common carotid artery and external carotid artery (ECA) were exposed through a midline neck incision. A silicon rubber-coated filament (Doccol Corporation, Sharon, MA, USA) was introduced into the left internal carotid artery (ICA) through the ECA stump to occlude the origin of the MCA. After a 60-min occlusion, reperfusion was achieved by filament withdrawal. The rectal temperature was maintained at 37 ± 0.5 °C. The heart rate and blood oxygen saturation level were monitored using a MouseOx system (STARR Life Science Corp., Oakmont, PA, USA). All recorded physiological parameters were maintained within normal physiological ranges (arterial O₂ saturation 91–98%, heart rate 350–410 bpm).

Stroke rats were randomly assigned to two groups: (1) phosphate-buffered saline (PBS) as a vehicle control; or (2) hUCB MNCs via intraarterial transplantation after reperfusion. In order to minimize the variability of MCAO, the exclusion criteria were that initial apparent diffusion coefficient (ADC) lesion volume at 30 min after MCAO was <100 mm³, indicative of incomplete occlusion. One rat from each group died during the follow-up study; the mortality rate (1/10) was equal in the two groups. Three rats from each group were excluded due to small initial ADC lesion. The final sample sizes were six rats for each group. The experimental design is shown in Fig. 1.

Timeline of the experiment. Phosphate-buffered saline (PBS) or human umbilical cord blood (hUCB) mononuclear cells (MNCs) were transplanted immediately after 60-min middle cerebral arterial occlusion (MCAO). Behavioral tests and magnetic resonance imaging (MRI) were performed at the indicated time points. ADC apparent diffusion coefficient, CBF cerebral blood flow, fMRI functional MRI, hMito human mitochondria, vWF von Willebrand factor