Transplanted Neural Stem Cells Promote Nerve Regeneration in Acute Peripheral Nerve Traction Injury: Assessment Using MRI

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1 Neuroradiology/Head and Neck Imaging Original Research Cheng et al. MRI of Peripheral Nerve Traction Injury Neuroradiology/Head and Neck Imaging Original Research Li-Na Cheng 1 Xiao-Hui Duan Xiao-Mei Zhong Ruo-Mi Guo Fang Zhang Cui-Ping Zhou Jun Shen Cheng LN, Duan XH, Zhong XM, et al. Keywords: cell labeling, MRI, neural stem cells (NSCs), peripheral nerve injury, stem cell transplantation DOI: /AJR Received August 14, 2010; accepted after revision October 15, Supported by grant from the National Natural Science Foundation of China and grant from the Natural Science Foundation of Guangdong Province. 1 All authors: Department of Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, No.107 Yanjiang Rd West, Guangzhou , Guangdong, China. Address correspondence to J. Shen (junshenjun@hotmail.com). AJR 2011; 196: X/11/ American Roentgen Ray Society Transplanted Neural Stem Cells Promote Nerve Regeneration in Acute Peripheral Nerve Traction Injury: Assessment Using MRI OBJECTIVE. The purpose of our study was to monitor neural stem cells (NSCs) transplanted in acute peripheral nerve traction injury and to use MRI to assess the ability of NSCs to promote nerve regeneration. MATERIALS AND METHODS. After labeling with gadolinium-diethylene triamine pentaacetic acid (gadopentetate dimeglumine) and fluorescent dye (PKH26), NSCs were grafted to acutely distracted sciatic nerves in 21 New Zealand White rabbits. In addition, unlabeled NSCs (n = 21) and vehicle alone (n = 21) subjects were injected as a control. Serial MRI was performed with a 1.5-T scanner to determine the distribution of grafted cells. Sequential T1 and T2 values of the nerves and functional recovery were measured over a 70- day follow-up period, with histologic assessments performed at regular intervals. RESULTS. The distribution and migration of labeled NSCs could be tracked with MRI until 10 days after transplantation. Compared with vehicle control, nerves grafted with labeled or unlabeled NSCs had better functional recovery and showed improved nerve regeneration but exhibited a sustained increase of T1 and T2 values during the phase of regeneration. CONCLUSION. Gadopentetate dimeglumine-based labeling allowed short-term in vivo MRI tracking of NSCs grafted in injured nerves. NSCs transplantation could promote nerve regeneration in acute peripheral nerve traction injury as shown by a prolonged increase of nerve T1 and T2 values. I njuries of the peripheral nerves are common and may lead to considerable disability and permanent neurologic deficits. Despite advancements in nerve repair by microsurgical techniques, the outcome of peripheral nerve injury, even after nerve repair, remains relatively poor. Recently, cellular therapy to repair injured nerves using Schwann cells, or stem and progenitor cells, has been shown to exert a beneficial effect on peripheral nerve regeneration and thus has been proposed as a new approach for functional reconstruction of the peripheral nerve injury [1]. In animal studies, embryonic neural stem cells (NSCs) exhibit regenerative success in repair of not only acutely transected nerves [2 4] but also chronically denervated peripheral nerves [5]. To guarantee the safety and maximum efficacy of these cellular therapies, a reliable method for in vivo monitoring of global cell distribution and cell dynamics is highly desirable, and careful investigation of the fate of transplanted cells is required [1]. MRI is the most attrac- tive imaging modality because it provides high-quality anatomic information with high soft-tissue contrast without ionizing radiation, which provides longitudinal follow-up of cell grafts and migration [6]. In CNS injury, ischemic stroke, and a variety of neurodegenerative diseases [7, 8], MRI had been successfully used to monitor the stem cell persistence and migration over time on cellular labeling techniques with gadoliniumbased T1-positvie [9] or iron-based T2-negative MR contrast agents [10]. However, few investigations have been conducted to explore the possibility of in vivo tracking of stem cells transplanted to treat acute peripheral nerve traction injury, whether NSCs could promote nerve regeneration in acute nerve traction injury, and whether this regenerative success could be detected by quantitative MRI. The purpose of this study was to investigate MRI tracking of NSCs after transplantation to the injured nerve on a simple gadopentetate dimeglumine-labeling technique and to assess the T1 and T2 values of the injured nerve over a 70- AJR:196, June

2 Cheng et al. day follow-up period after stem cell therapy in a rabbit model of acute peripheral nerve traction injury. Materials and Methods Animals All interventions and animal care procedures were performed in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (National Institutes of Health), and the Guidelines and Policies for Animal Surgery provided by our university and were approved by the institutional animal use and care committee. Sixty-three adult New Zealand White rabbits (weight range, 2 3 kg) were obtained from the animal experiment center of our university and were housed in a standard animal facility with 12-hour on-and-off light conditions and allowed standard food and water ad libitum. The anesthesia used in all cases consisted of an intraperitoneal injection of sodium pentobarbital (30 mg/kg of body weight). Cell Preparation NSCs were isolated and expanded from the prenatal brains of New Zealand White rabbits. The obtained NSCs were labeled in vitro by using Effectene (Qiagen) as the transfection reagent to transfer the standard MR contrast medium gadopentetate dimeglumine (Magnevist, Bayer Health- Care) [11, 12] into cells and followed by labeling with a fluorescent dye, PKH26. The detailed cell culturing, labeling procedure, resultant labeling feasibility, and efficiency have been described in our previous study [12]. Gadopentetate dimeglumine particles were present inside the cytoplasm of labeled cells, and there was no extracellular binding of gadopentetate dimeglumine to the cell membrane [12]. After labeling, cells were washed three times with Dulbecco s modified Eagle s medium/f12 by centrifugation (5 minutes, 300 g, 25 C) and were recovered in the culture medium. Before transplantation, cells were washed, counted, and resuspended in phosphate-buffered saline () to a final concentration of 10 5 cells/μl, and cell viability was determined to be greater than 90% through the trypan blue assay. Animal Surgery and Transplantation A Sunderland grade IV acute traction injury was created to one side of the sciatic nerve of each animal by one of the authors, who has 3 years of experience with microsurgical procedures, according to the previously reported method [13]. One week after injury, the 63 animals were randomly divided into three groups (21 in each group). The first group received labeled NSCs, the second group received unlabeled NSCs (i.e., untreated cells without gadopentetate dimeglumine or PKH26 labeling), and the third received as vehicle control. When grafting, the sciatic nerve was surgically exposed again, then a subepineurium injection of 5 μl of cell suspension or into the distracted portion of the sciatic nerve was performed using a 33-gauge needle attached to a 10-μl syringe at the rate of 1 μl/min. During injection, the needle was slowly advanced 5 mm proximally and then distally to allow uniform distribution of implanted cells. After injection, the needle was kept in place for 2 minutes before the needle was slowly withdrawn. MRI MRI was performed on a 1.5-T scanner (Intera, Philips Healthcare) with an 11-cm circular surface coil. The animals were placed in the lateral decubitus position after anesthesia and the hind limbs were positioned on the coil. The longitudinal images of the nerves were obtained in an oblique sagittal plane. To reveal signal abnormality, T1 and T2 values of the injured nerve after cell transplantation, 16 animals in each group underwent MRI and functional assessment before injury (baseline); immediately after transplantation at 1 week after injury; and 2, 3, 4, 6, 8, and 10 weeks after injury. The MR sequences included 3D fast spin-echo T2-weighted imaging with spectral fat suppression and 2D fast spin-echo T1-weighted imaging. T1 and T2 relaxation data were also acquired using a single-section mixed inversion recovery spin-echo sequence and a single-section multispin-echo sequence, respectively. Nerve T1 and T2 values were derived using the region of interest technique by one of the authors, who has 10 years of experience with musculoskeletal MRI, on the T1 and T2 maps calculated from T1 and T2 relaxation data. Each injured nerve was divided into three portions: proximal portion, distracted portion, and distal portions. The detailed MRI acquisition parameters and T1 and T2 measurements of individual portions of the injured nerve have been described previously [13]. After MRI and functional assessment, two animals each were scarified for histologic evaluation of nerve degeneration and regeneration at 2, 6, and 10 weeks after injury. To show the distribution and migration of the implanted cells, the other five animals in each group underwent MRI before and immediately after transplantation at 1 week after injury and 3, 7, 10, and 14 days after transplantation. The MR sequences included 3D fast spin-echo T1-weighted imaging with spectral fat suppression (TR/TE, 350/10; flip angle, 90 ; echo-train length, 6; section thickness, 2 mm; and overlapping gap, 1 mm) and 3D fast spin-echo T2-weighted imaging with spectral fat suppression (1600/80; flip angle, 90 ; echotrain length, 16; section thickness, 2 mm; and overlapping gap, 1 mm). Other acquisition parameters of both sequences were a field of view of mm, acquisition matrix of , image matrix of , and two signals acquired. In each group at each time, point one animal was sacrificed immediately after MRI for histologic assessment. Functional Assessment Functional recovery was assessed independently before MRI by two of the authors, each with 3 years of experience with this functional assessment, in blinded manner and by consensus. Functional assessment included observation of the presence of ankle dropping, semiquantitative toe-spreading reflex test, and the modified Tarlov score. An arbitrary 4-step scale (degree I IV) was applied to the toe-spreading index test, and a 5-step scale (grade 0 4) was applied to the modified Tarlov score test [13]. Histology To assess nerve regeneration, each tissue sample of three portions of the injured nerves harvested at 2, 6, and 10 weeks after the injury was divided into two parts. One part was fixed in 10% paraformaldehyde and sectioned longitudinally at 2-μm thickness and stained with standard H and E for light microscopy examination (TE2000-U, Nikon). The other part was fixed in 4% paraformaldehyde, dehydrated, osmificated, and embedded in plastic resin. Semithin sections of 0.5 μm were made and then stained with uranyl acetate and lead citrate for transmission electron microscopic examination (CM-10, Philips Healthcare). To assess the distribution and the fate of the implanted cells, samples of three portions of the injured nerves obtained immediately after injection and at 3, 7, 10, and 14 days after transplantation were processed for antineurofilament and anti-s100 immunofluorescence staining. Serial 6-μm thick longitudinal frozen sections were cut on a cryostat and rinsed twice with. Anti- Nestin (1:200, Millipore), anti-s100 (1:200, BD Pharmingen) and antineurodliament (1:200, BD Pharmingen) primary antibodies were applied to sections for 1 hour at room temperature (25 C). Then the sections were washed three times with, and FITC-conjugated secondary antibodies (1:200, BD Pharmingen) were applied for 30 minutes. The sections were examined and fluorescence images were obtained on an LSM 510 confocal microscope (Carl Zeiss) or a fluorescence microscope (TE2000-U, Nikon). Statistical Analysis T1 and T2 values were presented as means ± SD. One-way analysis of variance for repeated measures 1382 AJR:196, June 2011

3 MRI of Peripheral Nerve Traction Injury Fig. 1 In vivo MRI tracking of grafted neural stem cells. A C, On serial fat-suppressed T1-weighted images, labeled cells (arrows) showed subepineurium linear hyperintense signal immediately after injection in distracted portions (arrowheads). Afterward, this increased signal intensity gradually diminished and diffused into distracted and distal portions of injured nerve (A). However, reliable detection of labeled cells at spatial resolution of present experiment was no longer allowed by 14 days. No such high signal intensity could be found in nerve grafted with unlabeled cells (B) or phosphate-buffered saline (C). Inserted images in A are selectively zoomed pseudocolorized sciatic nerve images to better show high signal intensity of labeled cells in affected region of interest of by using OsiriX (open source) for MacIntosh (Apple) software. with the Student-Neuman-Keuls post-hoc test was used to analyze time-dependent and treatment-dependent observations of T1 and T2 values; one-way analysis of variance using Student-Newman-Keuls post-hoc analysis and the Student t test were used to compare T1 and T2 values between groups at single-experimental time points. Statistical significance was assigned at p < All statistical tests were performed by using SPSS 12.0 software. Results MRI and Histology of Transplanted Cells The grafted labeled cells were detected as a subepinerium linear hyperintense signal in the distracted portion of the injured nerves on T1-weighted imaging immediately after transplantation; thereafter, this high signal intensity gradually declined and diffused throughout the injured nerves from 3 days to 10 days after transplantation (Fig. 1), which was spatially parallel with the histologic distribution of labeled cells detected by fluorescence microscopy. Histologically, the labeled cells were initially located in the subepineurium around the injection site. Then, they gradually diffused along the epineurium distally and proximally and migrated deep into the nerve fascicles of the distracted portion, which reached a peak at 7 days after transplantation (Fig. 2). By 14 days, the labeled cells were no longer discernible on MRI, although they could be observed histologically. There was no high signal intensity or cellular red fluorescence found in the control groups (unlabeled and groups) during 14-day follow-up (Figs. 1 and 2). Immunofluorescence staining showed that the labeled cells were positive for Nestin at 3 days after transplantation, indicating survival of the grafted cells (Fig. 3). Fourteen days after transplantation, confocal images showed that a small amount of the labeled cells were located around regenerated axons and Schwann cells in the injured nerves, although no labeled cells were positive for neurofilament or S100 staining (Fig. 3). This indicated their lack of neuronal and glial differentiation. T1 and T2 Values of Nerves T1 and T2 values of nerves are shown in Figures 4 and 5. The proximal portions of the injured nerves in all three groups showed a slight increase in T1 and T2 values at 1 week after injury, followed by a rapid return to basal level at 2 weeks after injury. T1 values in the labeled group immediately after transplantation (1005 ± 48.3 milliseconds) were lower than those in unlabeled (1117 ± 50.2 milliseconds) and vehicle groups (1143 ± 49.1 milliseconds) (p = 0.036, 0.033). For the distracted and the distal portions of the injured nerves, T1 and T2 values in all groups reached a peak value at 1 week after injury; afterward, they began to slowly decrease but remained elevated at 10-week follow-up in the labeled and unlabeled groups. Fig. 2 Histologic distribution of grafted neural stem cells (NSCs). A E, Fluorescence images show that location of labeled cells was well correlated with MRI. NSCs with red fluorescence (arrows) were initially located around subepineurium injection site (A); afterward, they gradually diffused deep into nerve fascicles of distracted and distal portions (B D). Number of cells also gradually decreased over time; only small portion of cells was found by 14 days after transplantation (E). AJR:196, June

4 Cheng et al. T1 values of the distal portions in the vehicle group returned to near-normal levels at 4-week follow-up, whereas T1 and T2 values of the distracted and distal portions in the labeled and unlabeled groups remained higher than basal levels beginning at 4 weeks after injury. Notably, the distracted portions in the labeled group had lower T1 and T2 values (1370 ± 50.1 milliseconds and 88 ± 4.4 milliseconds) compared with those in the unlabeled group (1537 ± 57.8 milliseconds and T1 Values (ms) T2 Values (ms) * T1 Values (ms) T2 Values (ms) * A B C * * A B C T1 Values (ms) T2 Values (ms) Fig. 3 Histologic assessment of fate of grafted neural stem cells (NSCs). A D, Grafted NSCs with red fluorescence remained Nestin-positive at 3 days after transplantation (A and B). Confocal images show that small portion of grafted NSCs distributed along with neurofilamentpositive regenerated axon (C) and some NSCs dispersed among proliferated S100-positive Schwann cells (D) at 14 days after transplantation. 107 ± 4.9 milliseconds) and vehicle control (1526 ± 55.8 milliseconds and 112 ± 5.1 milliseconds) immediately after transplantation and a lower T1 (1119 ± 43.8 milliseconds) than those in the unlabeled group (1314 ± 47.8 milliseconds) and vehicle control group (1274 ± 47.4 milliseconds) at 2 weeks after injury (p = and p = 0.044). To observe the effect of cell transplantation on T1 and T2 values of the injured nerves, we compared T1 and T2 values between the unlabeled group and the group. T1 and T2 values in the labeled group were not used for analysis because the presence of gadopentetate dimeglumine or PHK26 in grafted labeled cells may interfere with T1 and T2 derivation from T1 and T2 pixel maps. In the distracted portions, T1 and T2 values in the unlabeled group (T1, 1126 ± ± 44.5 milliseconds; T2, 66 ± ± 3.5 milliseconds) were higher than those in the vehicle control group (T1, 1050 ± ± 30.3 milliseconds; T2, Fig. 4 Graphs show time course of T1 values. A C, Graphs show proximal portion (A), distracted portion (B), and distal portions (C) of nerves. NSCs = neural stem cells. Asterisk indicates p < 0.05 compared with unlabeled or phosphate-buffered saline () group, and indicates p < 0.05 compared with unlabeled group. Fig. 5 Graphs show time course of T2 values. A C, Graphs show proximal portion (A), distracted portion (B), and distal portions (C) of nerves. NSCs = neural stem cells. Asterisk indicates p < 0.05 compared with unlabeled or phosphate-buffered saline () group, and indicates p < 0.05 compared with unlabeled group AJR:196, June 2011

5 MRI of Peripheral Nerve Traction Injury 58 ± ± 3.8 milliseconds) during the period from 4 weeks to 10 weeks after injury (p = ). The distal portions in the unlabeled group had slightly higher T1 values (1055 ± ± 34.7 milliseconds) than those in the vehicle control group (956 ± ± 34.2 milliseconds) during the period from 3 weeks to 8 weeks after injury, whereas they had slightly higher T2 values (52 ± ± 3.0 milliseconds) than those in the vehicle control group (47 ± ± 3.1 milliseconds) during the period from 4 weeks to 10 weeks after injury (p = ). Nerve Histology Nerves in the three groups showed almost similar degeneration within 2 weeks after the injury. There was more pronounced Schwann cell proliferation and nerve fiber regeneration found in the distracted and distal portions in the labeled and unlabeled groups than in the vehicle control group at 6 weeks after injury. By 10 weeks after injury, nerves in both the labeled and unlabeled groups had more abundant and more mature regenerated axons with a thick myelin sheath than that of the control group (Fig. 6). Functional Recovery Functional assessment is shown in Figure 7. The labeled and unlabeled groups achieved better functional restoration compared with the vehicle control group. Ankle dropping in the three groups began to recover at 4 weeks after injury and was fully restored at 8 weeks after injury. However, there were more animals without ankle dropping in labeled (nine and 10, respectively) and unlabeled groups (six and nine, respectively) than in the vehicle control group (four and eight, respectively) at 4 and 6 weeks after injury. The labeled and unlabeled groups reached a higher grade of the toe-spreading reflex (1.83 ± ± 0 and 1.83 ± ± 0.29) than vehicle control (1.75 ± ± 0.62) and a higher Tarlov score (3.17 ± ± 0 and 3.08 ± ± 0) than vehicle control (3.00 ± ± 0) during the period from 4 weeks after injury to 10 weeks after injury. Fig. 6 Photomicrographs show histology of nerves after cell transplantation. A F, Histologic sections obtained from distracted portions of nerves were examined with light (A, C, and E) (H and E, 400) and electron microscopy (B, D, F) ( 3000). Nerves in all three groups showed similar axonal loss and myelin breakdown (arrows) at 2 weeks (left column) after surgery, but there was more prominent axonal and myelin regeneration (arrowheads) in nerves grafted with labeled (A and B) and unlabeled cells(c and D) compared with nerves grafted with phosphate-buffered saline (E and F) at 6 weeks (center column) and at 10 weeks (right column) after surgery. Discussion Many studies have established that NSCs can differentiate into Schwann-like cells, even neurons, and can promote axon regeneration after being transplanted into the transected peripheral nerve [2 5]. Tracking and monitoring these implanted stem cells using MRI can allow global dynamic evaluation of the distribution, migration, and fate of the cells noninvasively. To visualize these cells on MRI, T2-negative contrast agents (i.e., iron oxide nanoparticles) have been used more frequently than T1-positive contrast agents (gadolinium-based) considering the biocompatibility and higher imaging sensitivity of iron oxide particles. Nevertheless, tacking cells grafted to some tiny structures, such as the peripheral nerves, might not be beneficial from the iron oxide nanoparticles labeling technique because blooming susceptibility artifact inherent to the imaging would prevent clear visualization of the exact location and biodistribution of the grafted cells, and labeled cells in host tissue might be difficult to distinguish from microhemorrhage, micro air bubbles, or postsurgical abrasive iron-bearing material that could be introduced during direct injection AJR:196, June

6 Cheng et al. Toe-Spreading Reflex of stem cells [7]. Moreover, recent studies have shown that MRI of iron oxide labeled cells might be limited in the reliability of reporting long-term stem cell engraftment because the persistence of significant iron-dependent MRI signal could be derived from iron-containing tissue macrophages that may reuptake iron-oxide nanoparticles during the clearance of dead iron-labeled grafted cells after transplantation [14, 15]. In contrast, the pharmacologic properties of gadopentetate dimeglumine have been extensively investigated [16], and a previous pharmacokinetic study reported that gadopentetate dimeglumine in dead cells or interstitial spaces was washed out within 24 hours [17]. In this study, we labeled NSCs alternatively with paramagnetic gadopentetate dimeglumine to overcome the shortcomings potentially involved in using iron oxide particles. To validate the MRI findings, cells were colabeled with a cell membrane fluorescent dye (PKH26) with various advantages [18] to achieve histologic observation of the grafted cells. In this study, the distribution of grafted NSCs in the nerve could be clearly discerned on MRI until 10 days after transplantation. By 14 days after transplantation, the labeled NSCs could not be detected. The reason might be local dilution of contrast agent, which resulted from the absorption of gadopentetate dimeglumine released from the dead cells or continuous division and dispersion of viable labeled cells in the injured nerve. Moreover, NSCs could migrate deep into the fascicles of the injured nerve, indicating that the injury condition of the nerve might specifically attract grafted NSCs and promote their dispersion in the injured site. As revealed by confocal images, a small portion of grafted NSCs survived at 14-day Tarlov Score Fig. 7 Graph shows time courses of functional recovery for injured nerves. NSCs = neural stem cells, = phosphate-buffered saline. follow-up, and they could distribute along with regenerated axons and disperse among Schwann cells. Previous histologic investigation showed that NSCs or progenitor cells could survive and could differentiate into Schwann cells in a period of at least 6 weeks after transplantation [2 4]. However, the grafted viable cells were not found to achieve a neuronal or Schwann cell differentiation 2 weeks after transplantation in our study. The improved nerve repair for early cellular engraftment might be mostly paracrine because of neurotrophic factors. A similar finding was also reached in a study in which enhanced vascular regeneration into the ischemic environment occurred despite early death of injected progenitor cells in a mouse model of hind limb ischemia [15]. Prior work has shown that a native nerve with acute traction injury had prolonged T1 and T2 values, which peaked at 3 days after injury, and the T1 and T2 values and functional changes after injury showed a similar time course [13]. In this study, when transplanted with labeled NSCs, nerves with acute traction injury showed a transient decrease of T1 and T2 values during the period from immediately after transplantation to 2 weeks after transplantation, likely caused by the T1- and T2- shortening effect of the paramagnetic contrast agents loaded into grafted cells. T1 and T2 values in the distracted nerves and functional recovery after NSCs transplantation also showed a similar time course, whereas nerves grafted with labeled or unlabeled cells showed a relatively early and better functional recovery. Interestingly, they both showed more pronounced increase in T1 and T2 values than the vehicle control group during the phase of regeneration. Because histology confirmed that there were more pronounced Schwann cell proliferation and nerve fiber regeneration in the unlabeled and labeled NSCs groups during the phase of regeneration, a sustained increase in T1 and T2 values of the injured nerve after NSCs transplantation might be indicative of the enhanced nerve repair of stem cell transplantation. There were some limitations to our study. First, there was a relatively short duration (10 days after transplantation) of in vivo MRI tracking with the gadopentetate dimeglumine-labeling technique. The sensitivity of MRI with gadolinium compounds is low for cellular imaging purposes. MR detection thresholds in stem cell labeling were higher in the gadopentetate dimeglumine ( cells) than that of iron-containing particle ( cells) [16]. High gadolinium concentrations (greater than 10 4 M) are often necessary for major signal enhancement [16]. Moreover, the cell division or death of grafted cells would cause a progressive dilution of the amount of intracellular gadopentetate dimeglumine particles [19]. However, the gadolinium-based labeling technique in the current study is simple and could identify the labeled stem cells with high signal intensity on MRI. The increased signal intensity seen in the nerve correlated well with the true size of grafted stem cells, and the underlying nerve structures could be clearly discerned as well. Therefore, gadolinium-based labeling is still useful for cell tracking in a variety of experimental protocols, specifically, when local injection of cells is required to treat a small diseased structure. Second, whether gadopentetate dimeglumine particles loaded in labeled cells remained intracellular after transplantation was not determined. Histologic detection of gadopentetate dimeglumine could be achieved only by using the electron microscope. Using the similar gadopentetate dimeglumine labeling technique, our prior study showed that dead labeled cells could not produce high signal intensity even at 1 day after transplantation [20]. In this study grafted labeled cells showed a dynamic change of signal intensity over a 10-day period. Therefore, the possibility of having detected extracellular gadopentetate dimeglumine released from implanted cells was very low. From a research perspective, the development of reliable, sensitive, and nontoxic MR reporter genes could permit the unique long-term monitoring of living cells. There is significant interest and some progress in this field [21 23]. But these techniques are mostly limited to optical and 1386 AJR:196, June 2011

7 MRI of Peripheral Nerve Traction Injury PET imaging, and an MR reporter gene with a sensitivity sufficient for stem cell tracking is not available. In conclusion, short-term in vivo MRI tracking of the NSCs grafted to peripheral nerves with acute traction injury could be achieved using a simple gadopentetate dimeglumine-labeling technique. After transplantation into acutely distracted nerves, NSCs could promote nerve regeneration, and this enhanced nerve repair could be seen on MRI by a pronounced increase in the T1 and T2 values of the injured nerve during the phase of regeneration. References 1. Walsh S, Midha R. Practical considerations concerning the use of stem cells for peripheral nerve repair. Neurosurg Focus 2009; 26:E2 2. Aquino JB, Hjerling-Leffler J, Koltzenburg M, et al. In vitro and in vivo differentiation of boundary cap neural crest stem cells into mature Schwann cells. Exp Neurol 2006; 198: Murakami T, Fujimoto Y, Yasunaga Y, et al. Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve repair. Brain Res 2003; 974: MacDonald SC, Fleetwood IG, Hochman S, et al. Functional motor neurons differentiating from mouse multipotent spinal cord precursor cells in culture and after transplantation into transected sciatic nerve. J Neurosurg 2003; 98: Heine W, Conant K, Griffin JW, et al. Transplanted neural stem cells promote axonal regeneration through chronically denervated peripheral nerves. Exp Neurol 2004; 189: Bulte JW, Duncan ID, Frank JA. In vivo magnetic resonance tracking of magnetically labeled cells after transplantation. J Cereb Blood Flow Metab 2002; 22: Walczak P, Bulte JW. The role of noninvasive cellular imaging in developing cell-based therapies for neurodegenerative disorders. Neurodegener Dis 2007; 4: Syková E, Jendelová P. Magnetic resonance tracking of implanted adult and embryonic stem cells in injured brain and spinal cord. Ann N Y Acad Sci 2005; 1049: Modo M, Cash D, Mellodew K, et al. Tracking transplanted stem cell migration using bifunctional, contrast agent-enhanced, magnetic resonance imaging. Neuroimage 2002; 17: Guzman R, Uchida N, Bliss TM, et al. Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI. Proc Natl Acad Sci USA 2007; 104: Shyu WC, Chen CP, Lin SZ, et al. Efficient tracking of non-iron-labeled mesenchymal stem cells with serial MRI in chronic stroke rats. Stroke 2007; 38: Shen J, Cheng LN, Zhong XM, et al. Efficient in vitro labeling rabbit neural stem cell with paramagnetic Gd-DTPA and fluorescent substance. Eur J Radiol 2010; 75: Shen J, Zhou CP, Zhong XM, et al. MR neurography: T1 and T2 measurements in acute peripheral nerve traction injury in rabbits. Radiology 2010; 254: Terrovitis J, Stuber M, Youssef A, et al. Magnetic resonance imaging overestimates ferumoxide-labeled stem cell survival after transplantation in the heart. Circulation 2008; 117: Gianella A, Guerrini U, Tilenni M, et al. Magnetic resonance imaging of human endothelial progenitors reveals opposite effects on vascular and muscle regeneration into ischaemic tissues. Cardiovasc Res 2010; 85: Daldrup-Link HE, Rudelius M, Oostendorp RA, et al. Targeting of hematopoietic progenitor cells with MR contrast agents. Radiology 2003; 228: Ludemann L, Wurm R, Zimmer C. Pharmacokinetic modeling of GD-DTPA extravasation in brain tumors. Invest Radiol 2002; 37: Horan PK, Slezak SE. Stable cell membrane labeling. Nature 1989; 340: Walczak P, Kedziorek DA, Gilad AA, et al. Applicability and limitations of MR tracking of neural stem cells with asymmetric cell division and rapid turnover: the case of the shiverer dysmyelinated mouse brain. Magn Reson Med 2007; 58: Shen J, Zhong XM, Duan XH, et al. Magnetic resonance imaging of mesenchymal stem cells labeled with dual (MR and fluorescence) agents in rat spinal cord injury. Acad Radiol 2009; 16: Genove G, DeMarco U, Xu H, et al. A new transgene reporter for in vivo magnetic resonance imaging. Nat Med 2005; 11: Weissleder R, Moore A, Mahmood U, et al. In vivo magnetic resonance imaging of transgene expression. Nat Med 2000; 6: Gilad AA, McMahon MT, Walczak P, et al. Artificial reporter gene providing MRI contrast based on proton exchange. Nat Biotechnol 2007; 25: AJR:196, June