Phenotype of hUCB-MSCs

The cells picked from clones could be passaged successfully and the cells grew as a fusiform or fibroblast-like shape, whereas the subclones developed like a whirlpool (Figure 1).

Isolated hUCB-MSCs were characterized by flow cytometric analysis of specific surface antigens. hUCB-MSCs expressed surface antigens CD29 and CD44, but not CD34 and CD45, which was consistent with the surface antigens of bone marrow-derived MSCs (Figure 2).

hUCB-MSC transplantation affects NSSs and the infarct volume in MCAO rabbits

To evaluate the effect of hUCB-MSCs on neurological deficits after MCAO, we measured NSSs. The NSSs in the hUCB-MSC group at 7 and 14 days after reperfusion were less than those in MACO and saline groups (p < 0.05) (Figure 3).

Compared with MCAO and saline groups at 2 weeks after MCAO, infarct volumes in the hUCB-MSC group decreased remarkably (p < 0.05) (Figure 4).

hUCB-MSC transplantation affects neuronal metabolites in MCAO rabbits

To observe the effects of hUCB-MSC transplantation on neuronal metabolites in the infarct area, ¹H-MRS was used to detect the ratios of NAA/Cr, Cho/Cr ,and Lac/Cr at 24 h and 2 weeks after MCAO. Significant neuronal metabolic changes in the infarct area were observed at 24 h and 2 weeks after MCAO (Figure 5). ¹H-MRS spectra showed significant elevation in the Lac/Cr ratio and a marked decrease in NAA/Cr and Cho/Cr ratios at 24 h after MCAO in the MCAO group (p < 0.01 versus the sham group). Compared with saline and MCAO groups at 24 h after MCAO, NAA/Cr and Cho/Cr ratios were significantly increased and the Lac/Cr ratio was significantly decreased in the hUCB-MSC group (p < 0.01) (Figure 6). Interestingly, at 2 weeks after MCAO, NAA/Cr and Cho/Cr ratios had increased substantially in the hUCB-MSC group, whereas the Lac/Cr ratio has decreased remarkably compared with those in saline and MCAO groups (p < 0.01) (Figure 7).

Distribution and migration of hUCB-MSCs in the injured brain

To investigate whether transplanted hUCB-MSCs could migrate to the infarcted brain area, hUCB-MSCs labeled with 5-bromodeoxyuridine (BrdU) were injected via the right femoral vein of rabbits in the hUCB-MSC group. BrdU-positive cells migrated selectively to the infarction area, which were small with round or oval nuclei containing intensely stained brown chromatin (Figure 8).

hUCB-MSC transplantation affects neuronal density and OD of MAP2-positive cells in the infarct area

MCAO can cause a complete cerebral infarction as shown by magnetic resonance imaging (MRI) (Figure 9). Cell morphology in the sham group was normal with only a few necrotic cells. More necrotic cells were observed in MCAO and saline groups (Figure 10). MAP2 immunostaining products were localized within the cytodendrites of brain neurons in the sham group. MAP2 immunolabeling was markedly diminished in the infarct zone, and there was an abrupt transition between the infarct and adjacent normal tissue with occasional MAP2-positive cells in the transition zone (Figure 11). To investigate whether transplanted hUCB-MSCs could protect neurons, we examined neuronal density and OD of MAP2-positive cells in the infarct area. Neuronal density and OD of MAP2-positive cells in the MCAO group were significantly lower than those in the sham group (p < 0.05). However, neuronal density and OD of MAP2-positive cells in the hUCB-MSC group was higher than those in MCAO and saline groups (p < 0.05) (Figures 12 and 13). These data suggest that extensive neuronal damage occurs after MCAO and hUCB-MSC transplantation can prevent ischemic damage.

Effects of hUCB-MSC transplantation on CBCs and serum biochemical parameters in MCAO rabbits

Rabbit MCAO model

All animal protocols were approved by the Institutional Animal Care and Use Committee of Clinic College, Anhui Medical University. Male New Zealand white rabbits (provided by the Experimental Animal Center of Anhui Medical University, weighing 2.5–3.5 kg) were randomly divided into sham (n = 10), MCAO (n = 10), hUCB-MSC (n = 10), and saline (n = 10) groups. Anesthesia was induced with 2% sodium pentobarbital (20 mg/kg). The rectal temperature was maintained at 37°C throughout the surgical procedure as monitored by an electronic temperature controller linked to a heating pad (FHC, Bowdoinham, ME, USA). Transient MCAO was induced as described previously [25]. Briefly, the right common carotid artery (CCA), external carotid artery, and internal carotid artery (ICA) were exposed through a ventral midline incision. A nylon monofilament suture with a rounded tip (head-end diameter: 0.53 mm) was introduced into the CCA lumen and gently advanced into the ICA until it blocked the bifurcating origin of the MCA. At 2 h after occlusion, the rabbits were re-anesthetized and reperfused by withdrawing the suture until its tip cleared the lumen of the CCA.

hUCB-MSC culture and identification

Ethical approval for the use of hUCB-MSCs was obtained from Clinic College, Anhui Medical University. Human UCB samples (60–80 ml) were collected from the umbilical vein of full-term infants during deliveries with informed maternal consent. Isolation and expansion of hUCB-MSCs was conducted as reported previously [26]. Briefly, mononuclear cells were isolated by density gradient centrifugation. The separated mononuclear cells were washed, resuspended in Dulbecco’s modified Eagle’s medium, and then seeded at 1 × 10⁶ cells/mL. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂, and the culture medium was changed twice a week. Fibroblast-like adherent cells were observed in UCB-derived mononuclear cell cultures. After 1 –3 weeks, MSC colonies reached 80% confluence and the cells were trypsinized (0.25% trypsin), washed, resuspended in culture medium, and subcultured at 5 × 10⁴ cells/cm². MSCs of each hUCB harvest were expanded ex vivo by successive subcultivation under the same conditions. Passage 5–8 cells with a more than 1,000-fold expanding capacity were used in experiments.

Isolated hUCB-MSCs were characterized by flow cytometric analysis of specific surface antigens using CD29-FITC, CD45-PE, CD44-FITC, and CD34-PE monoclonal antibodies (BD Bioscience, San Jose, CA, USA). The cells were incubated in the dark at room temperature for 20 min.

Labeling of hUCB-MSCs with BrdU and tracing BrdU-positive cells in the injured brain

hUCB-MSCs at passage 5 were cultured in complete growth medium under the same conditions described above. Cells were labeled by incubation in medium containing 5 mmol/L BrdU (Roche Diagnostics, Mannheim, Germany) for 48 h before harvest. BrdU was added according to the manufacturer’s instructions. BrdU binds to DNA and labels the nucleus, serving as a tracer for in vivo studies of transplanted cells. The distribution of BrdU-positive cells was detected by immunohistochemical methods [27]. After 2 weeks, serial sections (6 μm thick) through the ischemic cortex were prepared from each rabbit. The sections were then incubated with a primary anti-BrdU monoclonal antibody (1:500) and then peroxidase-conjugated anti-rabbit IgG as the secondary antibody (1:100). The cells were stained with diaminobenzidine as the chromagen.

hUCB-MSC transplantation

Passage 5 hUCB-MSCs were injected intravenously within several minutes after MCAO. hUCB-MSCs (5 × 10⁶) diluted in 2 mL PBS were injected slowly for 5 min via the right leg femoral vein. For the saline group, 2 mL PBS without hUCB-MSCs was injected in the same manner.

Neurological deficit scale

Neurological deficits were measured in an independent and blinded fashion by NSSs as described by Purdy [28]. NSSs include motor function, consciousness, head turning, circling, and hemianopsia. The minimum NSS is 2, which is normal, and the maximal NSS is 11, indicating a comatose or dead animal. NSSs were measured at 6 h, 7 days, and 14 days after hUCB-MSC transplantation.

¹H-MRS examination

¹H-MRS data were obtained using a 1.5 Tesla MR machine (Siemens 1.5 T Novus MR scanner) and the point resolved echo spectroscopy method for localization and 5 mm × 5 mm × 5 mm volume of interest of the hippocampus in the bilateral temporal lobe. TE, TR, and NEX were 18, 400, and 200 ms, respectively. The ¹H-MRS detection technology used was multi-voxel spectroscopy sequence of fixed-point resolution data acquisition. All spectral data were automatically processed in probe sequence on the MR console. Probe acquisition protocols employed in this study included ratio frequency transmitter receiver gain adjustment, water suppression, auto-shim process, and data acquisition. The area of the peak for each organic compound and its comparative value were obtained for each volume of interest. Four major peaks were measured individually and determined to be Cho (3.2 ppm), Cr (3.0 ppm), NAA (2.0 ppm), and Lac (1.3 ppm), which were measured individually. Peak areas were expressed as the relative ratio to Cr in each spectrum. Cr was used as the reference because of its relative stability, even under rapid energy metabolism fluctuations [29]. Infarct volumes were automatically calculated with Tools Free-hand ROI software (Siemens).Two neuroradiologists analyzed all ¹H-MRS and MRI data.

Histopathological studies

At 2 weeks after MCAO, all rabbits were anesthetized with 20% urethane and then their brains were quickly removed and fixed with 10% formalin. After 24 h, the tissues were embedded in paraffin and serial sections (6 μm thick) were prepared through the ischemic cortex. The sections were then stained with hematoxylin and eosin. Neurons in a linear length (1 mm) were counted under a light microscope.

MAP2 immunohistochemistry

The sections were dewaxed with a standard procedure and washed with PBS as described previously [30]. Briefly, after blocking in 10% normal goat serum/PBS for 30 min at room temperature, the sections were incubated with a mouse anti-MAP-2 polyclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 16 h at 4°C. After three washes in PBS (10 min each), the sections were incubated with a goat anti-mouse secondary antibody for 2 h at room temperature. The sections were washed as described above and then incubated with an avidin biotinylated enzyme complex for 1 h at room temperature. The sections were visualized with diaminobenzidine. Nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated in an alcohol gradient and cleared with xylene. Ten images were then randomly captured in microscopic fields for each group. The images were used to analyze the OD of MAP2-positive cells with JEDA 801D software (Jiangsu JEDA Science Technology Development, China).

Statistical analysis

All data were expressed as the means ± standard error (SE). The Student’s t-test was used to compare differences between two groups. One-way analysis of variance was used to determine the significance of differences among multiple comparisons. Statistical significance was set at p < 0.05.