Abstract

BACKGROUND:

Noninvasive imaging techniques would be needed to validate the therapeutic benefits of cell transplantation therapy for central nervous system disorders.

OBJECTIVE:

To evaluate whether near-infrared (NIR)-emitting fluorescence tracer, quantum dots, would be useful to noninvasively visualize the bone marrow stromal cells (BMSC) transplanted into the infarct brain in living animals.

METHODS:

Rat BMSCs were labeled with QD800. In vitro and in vivo conditions to visualize NIR fluorescence were precisely optimized. The QD800-labeled BMSCs were stereotactically transplanted into the ipsilateral striatum of the rats subjected to permanent middle cerebral artery occlusion 7 days after the insult. Using the NIR fluorescence imaging technique, the behaviors of BMSCs were serially visualized during the 8 weeks after transplantation.

RESULTS:

NIR fluorescence imaging could noninvasively detect the NIR fluorescence emitted from the transplanted BMSCs engrafted in the peri-infarct neocortex through the scalp up to 8 weeks after transplantation. The intensity gradually increased and reached the peak at 4 weeks. The results were supported by the findings on ex vivo NIR fluorescence imaging and histological analysis.

CONCLUSION:

NIR fluorescence imaging is valuable in monitoring the behaviors of donor cells in the rodent brain. The results would allow new opportunities to develop noninvasive NIR fluorescence imaging as a modality to track the BMSCs transplanted into the brain.

Cell transplantation therapy has been expected to promote functional recovery in various kinds of central nervous system (CNS) disorders, including cerebral infarct. Embryonic stem cells, neural stem cells, induced pluripotent stem cells, and bone marrow stromal cells (BMSCs) are considered the candidates for donor cells. Of these, BMSCs are known to differentiate into neural cells.1,2 The BMSCs also support the host neurons and promote axonal regeneration by producing growth factors such as brain-derived neurotrophic factor.36 The BMSCs are far more accessible than other stem cells and pose no ethical and immunological problems because they can be obtained from the patients themselves.7 More important, they have no tumorigenesis.8,9 Several experimental studies show that transplanted BMSCs extensively migrated toward the peri-infarct area, expressed the phenotypes of neural cells, and improved neurological function in the rodent models of cerebral infarct.4,1013

Although these results are encouraging, several problems still remain unsolved before its clinical application. Most notably, it would be essential to develop the technique to track the transplanted cells in the host CNS serially and noninvasively to validate its beneficial effects. Several imaging approaches for tracking them have been proposed, including magnetic resonance imaging (MRI),1416 nuclear imaging,17 and optical imaging.1821 Of these, optical imaging has advantages including lower cost, rapid acquisition, no radiation toxicity, and relatively high sensitivity.7 Bioluminescence imaging provides high signal intensity, but requires the injection of high-dose luciferin and gene transfection.18,20 Fluorescence imaging has also been attempted in the field of cell transplantation therapy for CNS disorders, using green fluorescence protein (GFP). However, it is difficult to detect green fluorescence through the bone and skin because of its short wavelength.19,21,22 On the other hand, recent progress in nanotechnology has enabled us to apply biocompatible fluorescent semiconductor nanocrystals called quantum dots (QDs). The QDs have relatively narrow luminescence bands and high resistance to photobleaching.23 Near-infrared (NIR)–emitting QDs have much longer wavelengths that allow easy penetration of tissue, including bone and skin.24,25

This study aimed to evaluate whether NIR fluorescence tracer would be useful to noninvasively visualize BMSCs transplanted into the infarct brain in living animals.

MATERIALS AND METHODS

Preparation of BMSCs

All animal experiments were approved by the Animal Studies Ethical Committee of the Hokkaido University Graduate School of Medicine. The BMSCs were isolated under sterile conditions from 6- to 10- week-old Sprague-Dawley rats, as described previously.5,2628 The cells were passed 3 times for subsequent experiments.

Labeling of Cultured BMSCs With QDs

The cultured BMSCs were labeled with QD800 Q-tracker cell labeling kits (CdSeTe/ZnS: ∼10-nm core-shell type, 5 ∼10 nm polymer coating; Invitrogen, Hayward, California). Thus, 4 × 106 cells in 4-mL of Dulbecco modified Eagle medium containing 10% fetal bovine serum were cultured in a 25-cm2 tissue culture flask with 10 μL of QD800 reagent A and 10 μL of QD800 reagent B (5 nM). The cells were incubated at 37°C for 15 hours. After incubation, the cells were washed 3 times in phosphate-buffered saline. The NIR fluorescence emitted from the cultured BMSCs was visualized under an inverted fluorescence microscope (IX71; Olympus, Tokyo, Japan).

To determine optimal labeling condition, the cells were incubated under various conditions. The concentration of QDs ranged from 1 to 10 nM. Incubation time ranged from 1 to 15 hours. The labeling efficiency was analyzed by the fluorescence intensity emitted from QD800-labeled cell suspension (1.0 × 106 cells in 100 μL) on NIR fluorescence imaging (see later). The wavelengths of excitation light used were 710 nm. An emission filter of 800 nm was used.

Cell Viability Assay and Cell Growth Kinetics

The viability of a QD-labeled cell is essential for efficient in vivo cell tracking, but QDs per se have a potential cytotoxicity because QDs contains ZnS and Cd irons. The early phase cell viability after labeling with QDs was evaluated by using 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (TACS; R & D Systems, Minneapolis, Minnesota).29

After incubation of the BMSCs (1.0 × 105 cells/100 μL) with 0 (as control) to 10 nM QD800 for 1 to 15 hours in flat-bottomed 96-well plates, MTT (10 μL/well of a 5 mg/mL MTT stock solution in phosphate-buffered saline) was added to each well, and plates were incubated at 37°C for 4 hours. Subsequently, 100 μL of detergent was added to each well to dissolve the formed blue formazan. After all formazan was completely dissolved, the absorbance at 570 nm for the formazan solution was measured using the SUNRISE microplate reader (TECAN, Kawasaki, Japan). The survival rate of the cells was determined by calculating the ratio of absorbance of labeling group/absorbance of control group.

The late phase cell activity was evaluated by cell growth kinetics in subsequent culture after labeling with QD800. When grown to confluence, cells were detached with 0.05% trypsin-ethylenediamine tetraacetic acid (Gibco; Invitrogen), counted, and seeded at 1500 cells/cm2 to a 175-cm2 flask. Cumulative cell numbers were calculated as described previously.30

NIR Fluorescence Imaging

NIR fluorescence images were obtained using the IVIS 200 Imaging System (Xenogen Co., Alameda, California). All captured images were analyzed with the Living Image software (Xenogen Co.), and signal intensity was expressed in units of efficiency defined as efficiency (%) = emission light (photons/seconds)/excitation light (photons/seconds).

Visualization of QD800 in Rat Brain

To determine the optimal conditions to visualize the fluorescence emitted from QD800 in the rat brain, QD800 solution was directly injected into the rat brain. Burr holes were made 3 mm lateral to the bregma on both sides, using a small dental drill. A Hamilton syringe was inserted 2 mm into the brain parenchyma from the surface of the dura mater. Using an automatic microinjection pump (Model KDS-310; Muromachi Kikai Co., Tokyo, Japan), 10-μL solutions of QD800 (1000 nM and 500 nM) were injected in the left and right sides, respectively. After closure of the wound, the fluorescence emitted from QD800 in the brain was visualized, using the IVIS 200 Imaging System (Xenogen Co.). Excitation wavelengths used were 535, 600, 675, 710, and 745 nm. Regions of interest (ROIs)1 and 2 were placed on the injection sites, respectively. Region of interest 3 was placed on the occipital region as the reference. Then, total efficiency (TE) was calculated in each ROI. To determine optimal excitation wavelength that provided the clearest imaging with lowest autofluorescence of the skin, the target-to-normal ratio (TNR) was determined by calculating the ratio of TEROI1/TEROI3 and TEROI2/TEROI3. Exposure time was set to 5 seconds, and an 80-nm emission filter was used.

Next, to evaluate the number of transplanted cells that could be visualized on NIR fluorescence imaging, different amounts of QD800-labeled BMSCs (2.5 × 105 to 1.0 × 106) were injected 2 mm into the brain parenchyma from the dura mater, and to evaluate the correlation between the signal intensity and the depth from the surface of the dura mater, 1.0 × 106 cells were also injected 4 or 6 mm into the brain parenchyma from the surface of dura mater, then signal intensity was measured on NIR fluorescence imaging. The ROI was placed on the injection sites, and another ROI was placed on the contralateral occipital region as the reference, and the TE and TNR were then calculated. According to previous observations, the fluorescence emitted from QD800 was detected through the scalp and the cranium using an 800-nm emission filter and a 710-nm excitation filter.

Permanent Middle Cerebral Artery (MCA) Occlusion Model and BMSC Transplantation

Adult Sprague-Dawley rats were purchased from CLEA Japan, Inc (n = 20). Their body weight ranged from 280 to 350 g (mean body weight 326 g). A focal cerebral infarct was induced by permanent occlusion of the right MCA with temporary occlusion of the bilateral common carotid arteries (CCAs), as described previously with some modifications (n = 12).31 Anesthesia was induced by 4.0% isoflurane in N2O/O2 (70:30), and was maintained with 2.0% isoflurane in N2O/O2 (70:30). Core temperature was maintained at 36.5 to 37.0°C throughout the procedures. The bilateral CCAs were exposed through a ventral midline incision of the neck. Then, a 1.5-cm vertical skin incision was made between the right eye and ear. The temporal muscle was scraped from temporal bone, and a 5 × 5-mm temporal craniotomy was performed, using a small dental drill. To prevent cerebrospinal fluid leakage, the dura mater was carefully kept intact, and the right MCA was ligated using a 10-0 nylon thread through the dura mater. Then the cranial window was closed with the temporal bone flap. The temporal muscle and the skin were sutured with 4-0 nylon thread, respectively. Subsequently, the bilateral CCAs were occluded by surgical microclips for 1 hour. Only animals that circled toward the paretic side after surgery were included in this study.32 Sham-operated animals (n = 8) were also evaluated to elucidate the migratory capacity of the engrafted BMSCs. In the sham-operated group, all surgical procedures were performed as described, but the MCA and CCA were not occluded.

The QD800-labeled BMSCs were transplanted into the ipsilateral striatum at 7 days after the onset of ischemia (n = 20), as reported previously.12,13,21 A burr hole was made 3 mm right to the bregma, using a small dental drill. A Hamilton syringe was inserted 6 mm into the brain parenchyma from the surface of the dura mater, and 10 μL of cell suspension (1.0 × 106 cells) was introduced into the striatum over a period of 5 minutes, using an automatic microinjection pump. To prevent CSF leakage, the burr hole was covered with the pericranium, and the skin was sutured with 4-0 nylon thread.

In Vivo and Ex Vivo NIR Fluorescence Imaging of the QD800-Labeled BMSCs

In vivo NIR fluorescence imaging was repeated every week until 8 weeks after transplantation (n = 12 and 8 at 2 weeks after transplantation in MCA occlusion group and sham-operated group, respectively; n = 9 and 7 at 3 weeks; n = 7 and 7 at 4 weeks; n = 5 and 4 at 5 to 8 weeks). The animals were anesthetized as previously described and were shaved to avoid problems caused by light scattering because of the fur. They were then transferred to the chamber of the IVIS 200 Imaging System. The fluorescence emitted from the QD800 was detected through the scalp and the cranium, using an 800-nm emission filter and a 710-nm excitation filter. Exposure time was set at 5 seconds. On captured images, the ROIs were placed on the right parietal area and on the contralateral occipital region as the reference. The TE in each ROI was measured, and finally the TNR was calculated.

To validate the findings on in vivo NIR fluorescence images, the animals were killed at 2, 3, 4, and 8 weeks after transplantation (n = 3 and 1 at 2 weeks after transplantation in MCA occlusion group and sham-operated group, respectively; n = 2 and 0 at 3 weeks; n = 2 and 3 at 4 weeks; n = 5 and 4 at 8 weeks).12,13,21 The animals were transcardially perfused with 50 mL of heparinized saline under deep anesthesia, followed by 50 mL of 4% paraformaldehyde. Then, ex vivo NIR fluorescence imaging was performed using whole brain or 2-mm coronal brain slices.

Histological Analysis

The brain tissue was immersed in 4% paraformaldehyde for 2 days and embedded in paraffin. The 5-nm thick coronal sections were prepared for subsequent analysis. To localize QD800 in the brain, each section was observed through a 510-nm long-pass filter on a fluorescence microscope (BX51; Olympus).

To determine the fate of the transplanted BMSCs, fluorescence immunohistochemistry was performed at 8 weeks after transplantation.12,13,21 Each section was treated with the mouse monoclonal antibody against microtubule-associated protein 2 (dilution 1:200; Chemicon, Temecula, California), Tuj-1 (dilution 1:50, R & D Systems), or glial fibrillary acidic protein (dilution 1:500; BD Bioscience, Franklin Lakes, New Jersey) at 4°C overnight and treated with Zenon Alexa Fluor 488 (mouse IgG Labeling Kit; Molecular Probes Inc, Eugene, Oregon) at room temperature for 1 hour. The fluorescence emitted was observed through the appropriate filter on a fluorescence microscope.

Statistical Analysis

All data were expressed as mean ± standard deviation. Continuous data were compared by Student's t test or 1-factor analysis of variance followed by Dunnett's post hoc test as appropriate. Values of P < .05 were considered statistically significant.

RESULTS

Optimization of BMSC Labeling With QDs

Inverted fluorescence microscopy clearly showed that almost all cultured BMSCs were labeled with QD800. The NIR fluorescence emitted from QD800 could be observed as clear dots around the nucleus (Figure 1A-C).

FIGURE 1.

A-C, photomicrographs of rat bone marrow stromal cells (BMSCs) after incubation with QD800 showing that almost all cells are positive for QD800. Scale bar = 100 μm. QD, quantum dot. D, E, in vitro fluorescent images of the suspension (1.0 × 106 cells/100 μL phosphate buffered saline) of BMSCs incubated with different concentration of QDs ranging from 1 to 10 nM for different incubations times ranging from 1 to 15 hours, and their total efficiency (TE) measured by near-infrared fluorescence. F, the cell viability was examined by means of MTT assay. BMSCs were cultured in a 96-well plate at a density of 1 × 105 cells/well were incubated with different concentrations of QDs ranging from 1 to 10 nM for different incubation times ranging from 1 to 15 hours at 37°C. *P < .05, **P < .01. G, cumulative cell numbers of BMSCs after labeling with QDs. There were no differences in cell growth kinetics between QD-labeled cell and unlabeled BMSCs.

FIGURE 1.

A-C, photomicrographs of rat bone marrow stromal cells (BMSCs) after incubation with QD800 showing that almost all cells are positive for QD800. Scale bar = 100 μm. QD, quantum dot. D, E, in vitro fluorescent images of the suspension (1.0 × 106 cells/100 μL phosphate buffered saline) of BMSCs incubated with different concentration of QDs ranging from 1 to 10 nM for different incubations times ranging from 1 to 15 hours, and their total efficiency (TE) measured by near-infrared fluorescence. F, the cell viability was examined by means of MTT assay. BMSCs were cultured in a 96-well plate at a density of 1 × 105 cells/well were incubated with different concentrations of QDs ranging from 1 to 10 nM for different incubation times ranging from 1 to 15 hours at 37°C. *P < .05, **P < .01. G, cumulative cell numbers of BMSCs after labeling with QDs. There were no differences in cell growth kinetics between QD-labeled cell and unlabeled BMSCs.

When the BMSCs were incubated with QD800, the fluorescence signal intensity of the cell suspension was proportional to the QD800 concentration and to the incubation time. A higher concentration and longer incubation time yielded more intense NIR fluorescence (Figure 1D and E).

MTT assay revealed that QD800 did not affect the viability of BMSCs, when the concentration of QD800 was 5 nM or less and when the incubation time was shorter than 5 hours (Figure 1F). Based on these results, we labeled the BMSCs with 5 nM of QD800 for 15 hours for subsequent experiments. Under this condition, the QD800-labeled BMSCs exhibited normal proliferation capacity (Figure 1G).

In Vivo Imaging of QDs in Rat Brain

In vivo optical imaging could identify the NIR fluorescence emitted from QD800 injected into the brain through the cranium and scalp. When an emission filter was fixed at 800 nm, better spatial resolution was obtained with a longer excitation wavelength (Figure 2A-E). The highest TE was provided in both ROIs with an excitation wavelength of 675 nm (Figure 2G), but the highest TNR was obtained with an excitation wavelength of 710 nm (Figure 2H). Based on these observations, NIR fluorescence emitted from QD800 was detected in subsequent experiments, using an emission filter of 800 nm and excitation filter of 710 nm.

FIGURE 2.

In vivo fluorescence images of the rat that received a direct injection of QD800 into the brain, using excitation wavelengths of 535 nm (A), 600 nm (B), 675 nm (C), 710 nm (D), and 745 nm (E). Intracerebral QD800 can be visualized with better spatial resolution when the excitation wavelength was longer. When 3 regions of interest (ROIs) are set as shown in F, the excitation wavelength of 675 nm provides the highest total efficiency (TE) at both ROI1 and ROI2 (G). However, the highest target-to-normal ratio (TNR) is obtained under an excitation wavelength of 710 nm because of the lowest autofluorescence of the scalp (H).

FIGURE 2.

In vivo fluorescence images of the rat that received a direct injection of QD800 into the brain, using excitation wavelengths of 535 nm (A), 600 nm (B), 675 nm (C), 710 nm (D), and 745 nm (E). Intracerebral QD800 can be visualized with better spatial resolution when the excitation wavelength was longer. When 3 regions of interest (ROIs) are set as shown in F, the excitation wavelength of 675 nm provides the highest total efficiency (TE) at both ROI1 and ROI2 (G). However, the highest target-to-normal ratio (TNR) is obtained under an excitation wavelength of 710 nm because of the lowest autofluorescence of the scalp (H).

The QD800-labeled BMSCs were injected into the neocortex of nonoperated-on rats. The intensity of NIR fluorescence emitted from QD800-labeled BMSCs was proportional to their number and inversely proportional to their injection depth in the brain (Figure 3).

FIGURE 3.

The correlation between target-to-normal ratio (TNR) and grafted cell numbers or injection depth from the brain surface. Different amounts of QD-labeled bone marrow stromal cells ranging from 2.0 × 105 to 1.0 × 106 were injected 2 mm into the brain parenchyma from the dura mater (A-C, F). C-E, G, 1.0 × 106 cells were also injected 2 to 6 mm into the brain parenchyma from the dura mater.

FIGURE 3.

The correlation between target-to-normal ratio (TNR) and grafted cell numbers or injection depth from the brain surface. Different amounts of QD-labeled bone marrow stromal cells ranging from 2.0 × 105 to 1.0 × 106 were injected 2 mm into the brain parenchyma from the dura mater (A-C, F). C-E, G, 1.0 × 106 cells were also injected 2 to 6 mm into the brain parenchyma from the dura mater.

In Vivo and Ex Vivo NIR Fluorescence Imaging of the QD-Labeled BMSCs in the Permanent MCA Occlusion Model

In vivo NIR fluorescence imaging could not detect NIR fluorescence just after BMSC transplantation (Figure 4A). However, the NIR fluorescence could be serially visualized widely in the infarcted region through the cranium and scalp thereafter (n = 12). Thus, the intensity of NIR fluorescence signal gradually increased in the right parietal region during 4 weeks after transplantation. Subsequently, NIR fluorescence gradually decreased, but could be observed up to 8 weeks after transplantation (Figure 4B-G). Semiquantitative analysis revealed that the TNR gradually increased, reached a peak at 4 weeks, and gradually decreased thereafter (Figure 4O). On the other hand, the NIR fluorescence signal was identified at the injection site with much less intensity in the sham-operated animals (Figure 4H-N).

FIGURE 4.

Serial in vivo fluorescence images of the rat during 8 weeks after bone marrow stromal cell transplantation show that the near-infrared (NIR) fluorescence can be observed in the right parietal region between 1 and 8 weeks after transplantation in the middle cerebral artery occlusion group (A-G), but in sham-operated group, NIR fluorescence can be observed only at the site of injection, with low intensity (H-N). The column graph demonstrates that the total-to-normalized ratio in MCAO group is significantly higher at 1 to 8 weeks after transplantation than in the sham-operated group (O). *P < .01, **P < .001.

FIGURE 4.

Serial in vivo fluorescence images of the rat during 8 weeks after bone marrow stromal cell transplantation show that the near-infrared (NIR) fluorescence can be observed in the right parietal region between 1 and 8 weeks after transplantation in the middle cerebral artery occlusion group (A-G), but in sham-operated group, NIR fluorescence can be observed only at the site of injection, with low intensity (H-N). The column graph demonstrates that the total-to-normalized ratio in MCAO group is significantly higher at 1 to 8 weeks after transplantation than in the sham-operated group (O). *P < .01, **P < .001.

To localize the origin of NIR fluorescence, ex vivo NIR fluorescence imaging was performed at 2, 4, and 8 weeks after BMSC transplantation. Representative findings are shown in Figure 5. The NIR fluorescence detected on in vivo NIR fluorescence imaging (Figure 5A) could be visualized after the removal of the scalp (Figure 5B) and the cranium (Figure 5C), confirming that the NIR fluorescence was emitted from the brain. NIR fluorescence imaging of the coronal sections revealed that NIR fluorescence was emitted from the peri-infarct neocortex (Figure 5D), suggesting that the transplanted BMSCs aggressively migrated toward the peri-infarct area. However, in the sham-operated group, the NIR fluorescence was detected only at the injection track of transplantation (Figure 5E).

FIGURE 5.

Representative in vivo fluorescence image (A) and ex vivo fluorescence images after removal of scalp (B) and cranium (C) at 2 weeks after bone marrow stromal cell transplantation show that the near-infrared fluorescence (NIR) identified on in vivo imaging is emitted from the peri-infarct brain (arrows). Ex vivo fluorescence image (D, E) of coronal brain slices reveals that the NIR fluorescence is emitted from the peri-infarct neocortex in the occluded middle cerebral artery (MCAO), but fluorescence is emitted from only the site and tract of transplantation in sham-operated animals.

FIGURE 5.

Representative in vivo fluorescence image (A) and ex vivo fluorescence images after removal of scalp (B) and cranium (C) at 2 weeks after bone marrow stromal cell transplantation show that the near-infrared fluorescence (NIR) identified on in vivo imaging is emitted from the peri-infarct brain (arrows). Ex vivo fluorescence image (D, E) of coronal brain slices reveals that the NIR fluorescence is emitted from the peri-infarct neocortex in the occluded middle cerebral artery (MCAO), but fluorescence is emitted from only the site and tract of transplantation in sham-operated animals.

Histological Analysis

Fluorescence microscopy revealed that the NIR fluorescence emitted from the transplanted cells that migrated toward peri-infarct neocortex at different time points from 2 to 8 weeks after BMSC transplantation. The QD800-positive cells gradually spread to distribute widely in the peri-infarct area during 8 weeks after transplantation. The NIR fluorescence signals gradually attenuated during 8 weeks after transplantation (Figure 6).

FIGURE 6.

Ex vivo fluorescence image of coronal brain slices and photomicrographs on fluorescence microscopy at 2 (A-C), 4 (D-F), and 8 (G-I) weeks after bone marrow stromal cell transplantation in the middle cerebral artery occlusion (MCAO) group and the sham-operated group (J-L). On fluorescence microscopy, green indicates the autofluorescence of the host brain tissue as the background and red indicates fluorescence emitted from QD800. The QD800-positive cells aggressively migrate toward the cerebral infarct within 2 weeks after transplantation. The QD800-positive cells gradually were more widely distributed in the peri-infarct area. On the other hand, the QD800-positive cells could be detected only at the injection site and injection track at 8 weeks after transplantation in the sham-operated group. Scale bar = 200 μm.

FIGURE 6.

Ex vivo fluorescence image of coronal brain slices and photomicrographs on fluorescence microscopy at 2 (A-C), 4 (D-F), and 8 (G-I) weeks after bone marrow stromal cell transplantation in the middle cerebral artery occlusion (MCAO) group and the sham-operated group (J-L). On fluorescence microscopy, green indicates the autofluorescence of the host brain tissue as the background and red indicates fluorescence emitted from QD800. The QD800-positive cells aggressively migrate toward the cerebral infarct within 2 weeks after transplantation. The QD800-positive cells gradually were more widely distributed in the peri-infarct area. On the other hand, the QD800-positive cells could be detected only at the injection site and injection track at 8 weeks after transplantation in the sham-operated group. Scale bar = 200 μm.

Fluorescence immunohistochemistry revealed that a certain subgroup of QD800-positive cells were also positive for Tuj-1 and microtubule-associated protein 2 in the peri-infarct neocortex. Furthermore, some QD800-positive cells engrafted in the striatum were also positive for glial fibrillary acidic protein (Figure 7).

FIGURE 7.

Photomicrographs of fluorescence immunohistochemistry at 8 weeks after transplantation show that some of the quantum dot (QD)–positive cells are also positive for Tuj-1 (A-C) and glial fibrillary acidic protein (GFAP) (D-F) around the cerebral infarct (arrows). Scale bar = 50 μm.

FIGURE 7.

Photomicrographs of fluorescence immunohistochemistry at 8 weeks after transplantation show that some of the quantum dot (QD)–positive cells are also positive for Tuj-1 (A-C) and glial fibrillary acidic protein (GFAP) (D-F) around the cerebral infarct (arrows). Scale bar = 50 μm.

DISCUSSION

This study demonstrates that NIR-emitting QDs enable noninvasive tracking of BMSCs transplanted into the rat brain through the cranium and scalp for at least 8 weeks after transplantation, when the imaging protocol is optimized. This is the first report that notes the utility of NIR-emitting QDs in tracking the donor cells transplanted into infarct brain.

Labeling of BMSCs With NIR Fluorescent Tracer

To monitor the behaviors of the transplanted BMSCs, we used an exogenous cell labeling technique with NIR fluorescent tracer. This method has favorable features, including the need for only simple incubation with the dye according to standardized protocols and the capability to be applied to allogeneic or autologous cells without gene transfection. It also has the fundamental limitation of cell quantification and long-term monitoring because of photobleaching or degradation of fluorescent probe, dilution of tracer by cell proliferation or cell death, and possible transference to another cell such as a macrophage. Another problem is that persistence of fluorescent signals did not directly indicate the viability of transplanted cells. On the other hand, endogenous labeling such as luciferase reporter or GFP is free of these problems, but fluorescent protein in the NIR region is not available so far, and this method is unlikely to have human application because of the ethical problems and the possible side effects on cellular biological function because it requires gene transfection. In this study, the QDs were used to label the BMSCs because they are currently accepted as useful biological probes in view of their nanometer dimensions, attractive optical characteristics, high resistance to photobleaching or degradation, and strong fluorescence. Commercially available peptide-based QDs could be delivered into the cultured BMSCs in this study (Figure 1). Previous studies have shown that QDs may disturb cell growth and viability with a dose-dependent manner. In this study, however, labeling of the BMSCs with QD800 had minimal effects on their viability and proliferation capacity. Recent studies have shown that both embryonic stem cells and BMSCs can effectively be labeled with QDs.3336 QD labeling is also shown to have minimal effects on their viability, proliferation, and differentiation.3335

This study also shows that the intensity of NIR fluorescence detected largely depended on the wavelength of excitation light. Its intensity emitted from cell suspension was most prominent when excited by the light of shorter wavelength (data not shown). However, NIR fluorescence emitted from QD800 in the rat brain could be identified most clearly when excited at 710 nm, probably because excitation light with a shorter wavelength did not readily penetrate the brain through the cranium and scalp.

In Vivo Tracking of Transplanted BMSCs in Cerebral Infarct

We directly transplanted BMSCs into the ipsilateral striatum at 7 days after the onset of cerebral infarct for 3 reasons. First, it is not practical to transplant autologous BMSCs within 24 hours after the onset in a clinical situation because transplantation requires at least 1 week for the BMSCs harvested from the patients to expand. Second, a number of detrimental reactions are known to occur in the CNS tissue just after the onset, including circulatory disturbance, inflammatory responses, and the appearance of reactive oxygen species, excitotoxicity, and calcium overload.37 Finally, the BMSCs may interrupt the early self-repair system of the CNS by producing proteases such as matrix metalloproteases.6

Serial NIR fluorescence imaging reveals that the QD800-labeled BMSCs migrate into the peri-infarct area and can clearly be visualized through the cranium and scalp at 1 week after transplantation and that the highest NIR fluorescence signal is gained at 4 weeks after transplantation in the permanent MCA occlusion model. The findings on in vivo NIR fluorescence imaging are confirmed on ex vivo NIR fluorescence imaging and histological analysis and strongly suggest that this gradual signal increase in the ipsilateral parietal area results from the aggressive migration of transplanted QD800-labeled BMSCs. In this study, we used a sham-operated group as the control. However, in the sham-operated group, fluorescence signals detected on optical imaging were lower than those in the MCA occlusion group. It is most likely because the majority of engrafted BMSCs stayed in the injection site and did not migrate towards the peri-infarct area in the sham-operated group. Ex vivo optical imaging also supported this finding. Previous data have indicated that the SDF-1/CXCR4 system may play a critical role in both migration and proliferation of the transplanted BMSCs.13 As shown in Figure 7, some of transplanted BMSCs finally expressed neuronal marker and glial marker 8 weeks after transplantation, possibly because of differentiation or cell fusion, as reported previously.1,13,21,38

Previous reports have also revealed that the BMSCs migrate toward the peri-infarct area within 7 days when they are injected into the ipsilateral side22 and within 14 days when injected into the contralateral side.14,16,18 As demonstrated on histological analysis, these data also suggest that the transplanted BMSCs migrate toward the peri-infarct area within 14 days, but thereafter, some of them are more widely distributed around the peri-infarct area. Therefore, the qualitative signal intensity would gradually increase and reach the peak at 4 weeks after transplantation in this study.

Although the NIR fluorescence signal starts to decrease after 5 weeks after transplantation, it can be observed up to 8 weeks after transplantation. There are 3 possible explanations for the signal decrease between 5 and 8 weeks after transplantation. First, asymmetrical cell division and unequal division of endosomes to daughter cells may result in a dilution of QD800 labeling as they proliferate in the host brain. Yano et al38 reported similar results. Thus, they labeled the GFP-transgenic BMSCs with a superparamagnetic iron oxide and transplanted them into mice infarct brain. They found that the ratio of the superparamagnetic iron oxide to GFP-positive cells in the brain was approximately 2.7% at 3 months after transplantation. Second, their aggressive migration to the ventral side of infarct may cause it. Our ex vivo fluorescence imaging and histological analysis in Figure 6 support this speculation. Finally, the denaturation of QDs may largely contribute to the gradual attenuation of fluorescence emitted. Previously, Cai et al39 reported that QDs lost 14% of their original signal intensity after 24 hours of incubation in mouse serum. Histological findings in this study also showed that fluorescence signal emitted from QD800 was lower after 8 weeks after transplantation than after 2 and 4 weeks after transplantation.

We used direct injection of BMSCs because it may permit efficient delivery of the donor cells to the injured tissue. However, further investigations would be necessary to validate the different routes of cell delivery such as intravenous or intra-arterial injection.

Imaging Modalities

As aforementioned, the GFP-labeled cells could be visualized only through the mouse thin cranium or cranial window in previous experiments, thus being difficult to call a noninvasive technique.19,21,22 Previous studies reported that MRI can track the donor cells for almost the same period compared with this technique.15 This time period would be efficient to evaluate the engraftment and migration of transplanted cells. The findings suggest that in vivo NIR fluorescence imaging is comparable with MRI as a long-term, noninvasive imaging apparatus in the field of cell therapy for stroke. Therefore, NIR-emitting tracer would help to develop noninvasive optical imaging to track the transplanted cells in humans.

Previously, several studies evaluated the utility of NIR-emitting tracers for intravital imaging. Thus, Cy5.5 is known to emit fluorescence of maximally 694 nm. Several studies reported its utility to visualize pathophysiological conditions in the brain.40,41 Hintersteiner et al42 also synthesized NIR fluorescence dye that readily penetrates the blood-brain barrier and binds to amyloid plaque. They intravenously injected it in APP23 transgenic mice and identified NIR fluorescence emitted from the brain. Recently, Lin et al33 labeled embryonic stem cells with six different QDs and subcutaneously injected mice backs. They reported that QD800-labeled cells provided most prominent fluorescence intensity.33 Noh et al43 labeled the dendritic cells with QD800 and subcutaneously injected them in the mouse hindleg. They found that QD800 labeling had no adverse effects on the biological features of the dendritic cells and that in vivo optical imaging could track their homing into the lymph nodes.

Other imaging modalities such as MRI and nuclear imaging have no limit of penetration depth and can image the brain in 3 dimensions with better spatial resolution. A previous report indicated that nuclear imaging could detect even a single cell, and MRI could detect 100 cells when using high-field (17.6 T) MRI.44 But nuclear imaging is not appropriate for long-term monitoring, and MRI is sometimes impeded because of artifacts such as intracranial hemorrhage.45 Therefore, optical imaging might be an alternative or adjuvant modality in situations such as hemorrhagic infarction and traumatic brain injury. The current technique can visualize NIR fluorescence emitted from the BMSCs engrafted in the neocortex, but not in the striatum. The minimum number of the QD800-labeled cells that can be visualized in this technique may be approximately 2 × 105 cells, but when the cells are injected 6 mm deep from the brain surface, even 1 × 106 cells cannot be detected. In addition, optical imaging has lower spatial resolution compared with other modalities. The results strongly suggest the limitation of this technique to track the transplanted cells engrafted in the deep (more than several millimeters) regions of rat brain with good spatial resolution because of light scattering and absorption. Therefore, it would be essential to develop a biocompatible tracer with more intense NIR fluorescence and an imaging apparatus with more sensitive photon detectors to apply this modality in clinical situations. The QDs have a potential toxicity because of their heavy metal core, but their physicochemical properties are different when they are conjugated with different biomolecules. Thus, more biocompatible QDs are expected in future technology, which would enable introducing larger amounts of QDs into cells. The recent results are promising. Zhang et al46 indicated that the use of PEG-coated silanized QDs avoided the toxicity. Moreover, the advancement of imaging technology would enable detecting NIR fluorescence in the human brain surface with good time resolution.24,25

CONCLUSION

In vivo NIR fluorescence imaging is valuable in monitoring the behaviors of donor cells in the rodent brain. Furthermore, the current results open up new opportunities for developing noninvasive NIR fluorescence imaging as a modality to track the BMSCs transplanted into the human brain. Such techniques would be crucial to validate the therapeutic benefits of BMSC transplantation for CNS disorders.

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Acknowledgments

The authors thank Yumiko Shinohe for her technical assistance.

COMMENT

This is an interesting basic science research article detailing the use of near-infrared (NIR) fluorescence labeling with quantum dots (QDs) to track bone marrow stromal cells (BMSCs) that have been transplanted into the rat striatum 7 days post-infarction and is the first article to demonstrate the use of NIR-emitting QDs to track transplanted BMSCs in infarcted brain.

Research in the field of cell transplantation therapy for the treatment of central nervous system (CNS) disorders has grown significantly in the past decade. With continued advances in technology, cell transplantation therapy will become an increasingly important modality in the treatment of CNS disorders. BMSCs have significant advantages compared with other stem cells as they are readily obtained from the patients themselves, are known to differentiate into neural cells, support host neurons, and produce growth factors that facilitate axonal regeneration, as the authors note.

The authors demonstrate the safety and efficacy of NIR fluorescence labeling with QDs as a method of identifying and tracking BMSCs in a rodent model of cerebral infarction. QDs are fluorescent semiconductor nanocrystals that are relatively photostable and have narrow luminescence bands.1 Thus, compared with previous organic fluorophores, they can be observed longer. NIR-emitting QDs may be especially useful to track stem cells transplanted into the human brain because their longer wavelengths allow easier penetration of tissue such as bone and skin.

Other authors have already shown improved motor function after transplantation of BMSCs in a rat spinal cord injury model.2 The migration of BMSCs to the neocortex in the infarcted rat brains is an encouraging finding and supports the theory that BMSCs preferentially migrate to areas of infarcted brain. This is an exciting finding because this is the region in which the BMSCs can be most effective by differentiating into neural cells, supporting host neurons, or producing growth factors to facilitate axonal regeneration. The authors' finding that some of the transplanted BMSCs in the peri-infarct neocortex were positive for Tuj-1 and that some of the transplanted BMSCs in the corpus callosum were positive for glial fibrillary acidic protein demonstrate that these processes are actually occurring. Unfortunately, no information is available to demonstrate whether these findings translated into improved functional outcomes for the rats.

The authors' findings are extremely encouraging, but further studies are needed before these results can be translated into a clinical trial investigating the use of BMSCs for the treatment of ischemic stroke in humans.

Anitha Nimmagadda

Bernard R. Bendok

Chicago, Illinois

1.
Chiba Y, Kuroda S, Mariuchi K, et al. Transplanted bone marrow stromal cells promote axonal regeneration and improve motor function in a rat spinal cord injury model. Neurosurgery. 2009;64(5):911-999; discussion 999-1000.
2.
Michalet X, Pinaud FF, Bentolila LA, et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science. 2005;307(5709):538-544.

ABBREVIATIONS

    ABBREVIATIONS
  • BMSC

    bone marrow stromal cell;

  • CCA

    common carotid artery;

  • GFP

    green fluorescence protein;

  • MCA

    middle cerebral artery;

  • MTT

    3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide;

  • NIR

    near-infrared;

  • QD

    quantum dot;

  • TE

    total efficiency;

  • TNR

    target-to-normal ratio