Male Sprague-Dawley rats, weighing 200–250 g, were obtained from the Experimental Animal Centre of China Medical University, Shenyang, China. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of China Medical University. All animals were housed under diurnal lighting conditions (22±3°C and a 12 h light/dark cycle) with free access to standard laboratory chow and water.

Isolation and ex vivo expansion of EPCs were performed as previously described (19,20). Briefly, femur and tibias were excised from the donor rats after they were anesthetized with chloral hydrate (300 mg/kg) and sacrificed by cervical dislocation. Total bone marrow cells were harvested aseptically by flushing femurs and tibias with phosphate buffered saline (PBS), and mononuclear cells were collected by density gradient centrifugation with Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA). After washing twice in PBS, the obtained mononuclear cells were seeded on rat plasma fibronectin-coated dishes and maintained in EC basal medium-2 (EBM-2) supplemented with 5% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), antibiotics, and growth factors. Cells were cultured at 37°C in a humidified atmosphere with 5% CO₂. After 4 days of culture, the non-adherent cells were removed by washing with PBS, and fresh culture medium was applied. On the tenth day, the adherent cells were harvested by trypsinization, washed, and resuspended for transplantation or immunophenotypic characterization by flow cytometry.

To characterize the EPC population, flow cytometry was performed on freshly isolated cells (at Day 0) and on cells after 10 days of culture by using the antibodies against a panel of surface markers, including CD31, CD34, CD133 and fetal liver kinase-1 (Flk-1). Briefly, the cells were harvested, washed twice with PBS, and then incubated for 30 min at 4°C with mouse anti-rat antibodies for CD31 and CD34 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and rabbit anti-rat antibodies for CD133 and Flk-1 (Abnova, Taipei, Taiwan), followed by their corresponding fluorescein isothiocyanate (FITC)-labeled secondary antibodies. Finally, the samples were analyzed with a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA) and data were processed using the CellQuest software program.

In order to track the fate of transplanted cells, EPCs after 10 days of culture were labeled with a lipophilic red fluorescent dye, CM-Dil (Invitrogen), according to the manufacturer’s instructions. Thirty minutes before transplantation, EPCs were incubated with 2 μl CM-Dil (1 mg/ml) at 37°C and then resuspended in 500 μl culture medium for administration.

Fifty-four rats were randomly divided into three groups as follows (18 rats in each group): the sham group, without MCAO and culture medium-treated; the I/R group, 2 h occlusion followed by 24 h reperfusion and culture medium-treated; the EPC group, with MCAO and EPC administration.

A rat model of cerebral I/R injury was established by MCAO as previously reported (21). In brief, after the rats were anesthetized with 10% chloral hydrate (350 mg/kg, i.p.), the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were surgically exposed through a ventral middle line incision. A 4-0 nylon monofilament nylon suture with a heat-rounded tip was introduced into the ECA lumen and gently advanced into the ICA lumen until a slight resistance was encountered, thus to occlude the origin of the middle cerebral artery. Two hours later, the filament was carefully withdrawn to restore cerebral blood flow and the neck incision was sutured. The rectal temperature was maintained between 36.5 and 37.5°C by using a heat pad and a heat lamp throughout the surgical procedure. Sham-operated animals underwent the same surgical procedures but without inserting a filament.

Labeled EPCs (10⁶ cells in 500 μl culture medium) were infused into the rats in the EPC group at the onset of reperfusion and 12 h after reperfusion via the tail vein. Rats in the sham and I/R groups were injected in the same manner but only with an equal volume of fresh culture medium. After 24 h of reperfusion, all animals were deeply anesthetized with chloral hydrate (300 mg/kg) and sacrificed by decapitation.

After decapitation, the brains (n=6) were quickly collected, chilled in ice-cold saline for 5 min, and sectioned coronally into eight, 1.5 mm slices. The slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich) at 37°C for 20 min, followed by fixation in 4% paraformaldehyde overnight. The normal tissues were stained dark red, while the infarct areas remain unstained (white). The stained slices were photographed with a digital camera, and the infarct area was quantified with an image analysis system (Image-Pro Plus). The infarct volumes were presented as percentages of the total brain volume (22).

Antioxidative enzyme activities including SOD, GSH-PX, GSH and MDA in the hippocampus homogenate were measured by using commercially available kits (Jiancheng Institute of Bioengineering, Nanjing, China), according to the recommended protocols. SOD activity was determined by monitoring its ability to inhibit nucleotide oxidation. Results were reported as U/mg protein. A GSH-PX unit activity was defined as the amount of enzyme required to convert 1 μmol of reduced GSH to the oxidized form of glutathione GSH in the presence of H₂O₂/min. MDA content was determined based on its activity to react with thiobarbituric acid and is expressed as nmol/mg protein. The activity of caspase-3 in the hippocampus homogenate was assayed by using a caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, P.R. China) following the manufacturer’s instructions. Caspase-3 activity was estimated by measuring Ac-DEVD-pNA reacting substances at the wavelength of 405 nm.

The brains were immersed in 10% buffered formalin overnight, embedded in paraffin, and sectioned with a thickness of 5 μm. After dewaxing with xylene and rehydrating through descending ethanol, the sections were subjected to heat-mediated antigen retrieval in citrate buffer solution (pH 6.0). Subsequently, endogenous peroxidase activity was inactivated using 3% (v/v) hydrogen peroxide at room temperature for 15 min, and nonspecific binding of antibodies was blocked with 10% normal goat serum. The sections were then incubated overnight at 4°C in a humidifying chamber with a rabbit anti-rat NF-κB polyclonal antibody (1:200 diluted; Santa Cruz Biotechnology, Inc.). After rinsing in PBS, the corresponding secondary antibody [biotinylated goat anti-rabbit immunoglobulin G (IgG), 1:200 diluted; Zhongshan Golden Bridge Biotechnology, Beijing, China] was applied, followed by incubation for 30 min with streptavidin-horseradish peroxidase conjugate. The final immunoreactivity was visualized by using 3,3′-diaminobenzidine (DAB), and all sections were then counterstained with hematoxylin, dehydrated, and mounted. For negative controls, an isotype matched IgG was used instead of the primary antibody.

Total proteins were extracted from the ipsilateral ischemic cortex, and protein concentrations were quantified using bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). An equivalent amount of proteins was resolved on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), and then electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) under 70 V constant voltage conditions for 90 min. After blocking with 5% fat-free milk at room temperature for 2 h, the membranes were immunoblotted overnight at 4°C with rabbit anti-Bcl-2, anti-Bax, (1:1,000 diluted; Abcam, Cambridge, MA, USA), or anti-NF-κB antibody (1:1,000 diluted; Santa Cruz Biotechnology, Inc.), followed by horseradish peroxidase-conjugated secondary antibodies. Following extensive washing, immunoblots were visualized with an enhanced chemiluminescence (ECL) detection kit (Millipore). The intensity of the bands was quantified by densitometric analysis and normalized to the loading control β-actin.

All data are expressed as the means ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA followed by the Bonferroni post-hoc test for individual comparisons between group means. Graphs were plotted and statistical calculations were carried out using SigmaPlot 12.0 (Systat Software, Inc., San Jose, CA, USA). A P-value <0.05 was considered to indicate statistically significant differences.

After 10 days of culture in EC basal medium, the bone marrow-derived mononuclear cells exhibited spindle-shaped morphology or cobblestone-like appearance typical of endothelial cells (Fig. 1A). To further characterize these cells, the stem cell markers (CD34 and CD133) and endothelial cell markers (CD31 and Flk-1) were analyzed by flow cytometry according to previous studies (23,24). Compared with freshly isolated mononuclear cells, the percentages of cell population positive for CD34, CD133, CD31 and Flk-1 were increased from 22.30±2.56 to 70.81±4.89%, 19.5±2.95 to 81.66±5.33%, 10.83±1.12 to 46.81±3.54% and 12.94±1.87 to 60.05±3.91%, respectively (Fig. 1B). These cells were therefore confirmed as bone marrow-derived EPCs.

To investigate the neuroprotective effects of EPC transplantation on cerebral I/R injury, EPCs (10⁶ cells) were administered intravenously at the onset of reperfusion and 12 h after reperfusion, and infarct volumes were assessed at 24 h after reperfusion by TTC staining. As shown in Fig. 2A and B, no infarction was observed in sham-operated group animals. By contrast, the infarct volume was 28.9±1.8% after 2 h MCAO and 24 h reperfusion in the I/R group. However, EPC transplantation significantly reduced cerebral infarct volumes, indicating neuroprotective effects of EPC transplantation against cerebral I/R injury. Moreover, under immunofluorescence microscopy, a significant number of CM-Dil-labeled EPCs showing red fluorescence adoptively transferred into the infarct area, suggesting that EPCs can migrate into the ischemic rat brain within 24 h after intravenous injection.

To test if EPC transplantation exerts its neuroprotective effects against cerebral I/R injury via the inhibition of neuronal apoptosis, we detected the expression of Bcl-2 and Bax protein in the ischemic penumbra of rats with 2 h MCAO and 24 h reperfusion by western blot analysis. The results showed that cerebral I/R injury induced by MCAO caused significant upregulation of Bax and downregulation of Bcl-2 protein levels; however, these changes were remarkably attenuated by EPC transplantation (P<0.05, n=6) (Fig. 3A and B). We also measured the enzymatic activity of caspase-3, a reliable indicator for assessment of apoptosis. The activity of caspase-3 in the ischemic penumbra of rats with 2 h MCAO and 24 h reperfusion was significantly increased compared with the sham group, while the caspase-3 activity was markedly reduced in the EPC group (P<0.05, n=6) (Fig. 3C). These findings suggest that EPC transplantation has the potential to protect hippocampal neurons against cerebral I/R injury-induced apoptosis.

To further determine whether the neuroprotective effects of EPC transplantation are associated with the inhibition of oxidative stress, we evaluated the content of MDA and the activities of antioxidant enzymes in the ischemic penumbra of rats with 2 h MCAO and 24 h reperfusion. As illustrated in Fig. 4A, the level of MDA, an index of lipid peroxidation, was significantly increased in culture medium-treated ischemic hippocampus from 0.61±0.09 to 2.19±0.32 nmol/mg protein (P<0.01, n=6). After administration of EPCs, the I/R-induced lipid peroxidation was evidently decreased to 1.37±0.17 nmol/mg protein (P<0.01, n=6) compared to the I/R group. Additionally, the activities of antioxidant enzymes including GSH, GSH-PX and SOD in the I/R group were remarkably diminished compared to the sham group (P<0.01, n=6). However, EPC transplantation resulted in enhanced activities compared to the I/R group (P<0.05, n=6). Collectively, our data indicate that administration of EPCs exerts neuroprotective effects via its ability to inhibit oxidative stress.

Next, we analyzed the effects of EPC transplantation on NF-κB expression in hippocampus after I/R injury by immunohistochemistry and western blot analysis. Few NF-κB positive cells were observed in the hippocampus of sham-operated animals (Fig. 5A). At 24 h after reperfusion, an intense staining of NF-κB was found in the nucleus in the ischemic hippocampus. However, EPC implantation significantly attenuated the rise of brain I/R-induced NF-κB expression. Meanwhile, changes observed by western blot analysis were in accordance with the observations in the immunohistochemical staining (Fig. 5B and C). These findings demonstrate that EPC transplantation is capable of inhibiting brain I/R injury-induced NF-κB activation.