MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain.

Stem cell transplantation is being explored as a new paradigm for the treatment of demyelinating diseases. Magnetically labeled multipotential neural precursor cells were transplanted into the ventricles of rats with acute experimental allergic encephalomyelitis (EAE) and high-resolution (microscopic) MR images were obtained ex vivo. Migration patterns of live cells into periventricular white matter structures could be easily visualized, with a good correlation of the corresponding histopathology. The present results confirm that MR cell tracking can be used to guide the development of successful transplantation protocols.     

大鼠间充质干细胞超顺磁性氧化铁标记和经脾移植后的MR示踪研究

目的磁粒子标记骨髓间充质干细胞,同种异体移植入急性肝损伤大鼠脾脏,应用1·5TMR仪进行活体成像,为干细胞移植并无创示踪提供实验依据。方法制备Fe2O3-多聚左旋赖氨酸(PLL),标记分离纯化后的大鼠骨髓间充质干细胞(bonemesenchymalstemcells,BMSCs),普鲁士蓝染色显示细胞内铁。12只急性肝损伤大鼠分成实验和对照两组,将标记细胞(实验组,n=6)和未标记细胞(对照组,n=6)同种异体移植入大鼠脾脏,在移植前、移植后3h及3、7、14d应用MR的T2W对肝脏进行活体成像,测量肝脏信噪比(signal-to-noiseratio,SNR),并与肝脏组织切片普鲁士蓝染色对照。结果BMSCs的Fe2O3-PLL标记率近100%,普鲁士蓝染色显示蓝色铁颗粒位于BMSCs胞质内。实验组和对照组注射前、注射后3h及3、7、14d肝脏的SNR分别为19·53±2·30,3·28±1·06,7·34±2·10,10·25±3·96,15·50±3·73;20·20±4·35,21·20±4·43,19·13±2·80,21·43±5·45,19·07±4·80。与注射前比较,实验组3h及3、7d肝脏的SNR下降,差异具有统计学意义(Dunnett检验,t值分别为16·25,12·19,9·29,P值均<0·05),14d时SNR虽仍较低,但差异无统计学意义(Dunnett检验,t=4·03,P>0·05)。对照组移植后各时间点与移植前比较,SNR改变差异无统计学意义(方差分析,F=0·187,P>0·05)。组织学示普鲁士蓝阳性细胞主要分布于肝小叶中央静脉周围病变区。结论Fe2O3-PLL可以有效标记大鼠BMSCs,临床应用型1·5TMR仪可对磁粒子标记的BMSCs经脾植入后进行活体示踪。     

Dual in vivo magnetic resonance evaluation of magnetically labeled mouse embryonic stem cells and cardiac function at 1.5 t.

Cell therapy has demonstrated the potential to restore injured myocardium. A reliable in vivo imaging method to localize transplanted cells and monitor their restorative effects will enable a systematic investigation of this therapeutic modality. The dual MRI capability of imaging both magnetically labeled mouse embryonic stem cells (mESC) and their restorative effects on cardiac function in a murine model of acute myocardial infarction is demonstrated. Serial in vivo MR detection of transplanted mESC and monitoring of the mESC-treated myocardium was conducted over a 4-week period using a 1.5 T clinical scanner. During the 4-week duration, the mESC-treated myocardium demonstrated sustained improvement of the left ventricular (LV) ejection fraction and conservation of LV mass. Furthermore, no significant difference of their restorative effects on the cardiac function was created by the magnetic labeling of mESC. Thus, in vivo MRI enables simultaneous detection of transplanted mESC and their therapeutic effect on the injured myocardium.     

Long‐term MR cell tracking of neural stem cells grafted in immunocompetent versus immunodeficient mice reveals distinct differences in contrast between live and dead cells

Neural stem cell (NSC)‐based therapy is actively being pursued in preclinical and clinical disease models. Magnetic resonance imaging (MRI) cell tracking promises to optimize current cell transplantation paradigms, however, it is limited by dilution of contrast agent during cellular proliferation, transfer of label from dying cells to surrounding endogenous host cells, and/or biodegradation of the label. Here, we evaluated the applicability of magnetic resonance imaging for long‐term tracking of transplanted neural stem cells labeled with superparamagnetic iron oxide and transfected with the bioluminescence reporter gene luciferase. Mouse neural stem cells were transplanted into immunodeficient, graft‐accepting Rag2 mice or immunocompetent, graft‐rejecting Balb/c mice. Hypointense voxel signals and bioluminescence were monitored over a period of 93 days. Unexpectedly, in mice that rejected the cells, the hypointense MR signal persisted throughout the entire time‐course, whereas in the nonrejecting mice, the contrast cleared at a faster rate. In immunocompetent, graft‐rejecting Balb/c mice, infiltrating leukocytes, and microglia were found surrounding dead cells and internalizing superparamagnetic iron oxide clusters. The present results indicate that live cell proliferation and associated label dilution may dominate contrast clearance as compared with cell death and subsequent transfer and retention of superparamagnetic iron oxide within phagocytes and brain interstitium. Thus, interpretation of signal changes during long‐term MR cell tracking is complex and requires caution. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.     

超顺磁性氧化铁标记大鼠骨髓基质细胞经门静脉移植MRI活体示踪的研究

目的观察1．5T场强MRI联合动物专用线圈是否可以活体示踪经门静脉移植的纳米级超顺磁性氧化铁颗粒标记的骨髓基质细胞（bone marrow stromal cells BMSCs），为介人性门静脉骨髓基质细胞移植治疗终末期肝脏疾病的研究提供进一步的依据。方法供体大鼠5只，梯度密度离心分离BMSCs，纳米级超顺磁性氧化铁颗粒和脂质体转染BMSCs，体外经普鲁士蓝染色和HE染色确定细胞标记率。受体大鼠15只，分为5组，分别为对照组和纳米级超顺磁性氧化铁颗粒标记的骨髓基质细胞经门静脉移植人正常大鼠肝脏后2h、3d、7d及2周组。1．5T场强MRI联合动物专用线圈行T1W、T2W和T2＊序列扫描，观察肝脏信号改变情况，与对照组比较，并且与组织切片对照。结果纳米级超顺磁性氧化铁颗粒和脂质体转染BMSCs，细胞标记率〉95％。经门静脉移植人正常大鼠肝脏后，T2＊序列扫描显示经标记的BMSCs在肝内显示弥漫性的结节性低信号影，移植后2h到2周均可见到细胞在受体肝脏内存在，组织学切片显示信号缺失部位与铁颗粒标记细胞相一致。结论纳米级超顺磁性铁氧体颗粒标记的大鼠BMSCs经门静脉移植后可以通过1、5T场强行MRI活体示踪，为临床干细胞移植的应用提供可行的示踪方法。     

In vivo MR imaging tracking of transplanted mesenchymal stem cells in a rabbit model of acute peripheral nerve traction injury

Purpose

To investigate in vivo MRI tracking mesenchymal stem cells (MSCs) in peripheral nerve injures using a clinically available paramagnetic contrast agent (Gd‐DTPA) and commercially available rhodamine‐incorporated transfection reagents (PEI‐FluoR).

Materials and Methods

After bone marrow MSCs were labeled with Gd‐DTPA and PEI‐FluoR complex, the labeling efficacy and longevity of Gd‐DTPA maintenance were measured and cell viability, proliferation, and apoptosis were assessed. Thirty‐six rabbits with acute sciatic nerve traction injury randomly received 1 × 10⁶ labeled (n = 12) or unlabeled MSCs (n = 12) or vehicle alone injection. The distribution and migration of implanted cells was followed by MRI and correlated with histology. The relative signal intensity (RSL) of the grafts was measured.

Results

The labeling efficiency was 76 ± 4.7% and the labeling procedure did not in?uence cell viability, proliferation, and apoptosis. A persistent higher RSL in grafts was found in the labeled group compared with the unlabeled and vehicle groups until 10 days after transplantation (P < 0.05). The distribution and migration of labeled cells could be tracked by MRI until 10 days after transplantation. Transplanted MSCs were not found to transdifferentiate into Schwann‐like cells within 14‐day follow‐up.

Conclusion

Labeling MSCs with the dual agents may enable cellular MRI of the engraftment in the experimental peripheral nerve injury. J. Magn. Reson. Imaging 2010;32:1076–1085. © 2010 Wiley‐Liss, Inc.

     

Neural precursors exhibit distinctly different patterns of cell migration upon transplantation during either the acute or chronic phase of EAE: A serial MR imaging study

As the complex pathogenesis of multiple sclerosis contributes to spatiotemporal variations in the trophic micromilieu of the central nervous system, the optimal intervention period for cell‐replacement therapy must be systematically defined. We applied serial, 3D high‐resolution magnetic resonance imaging to transplanted neural precursor cells (NPCs) labeled with superparamagnetic iron oxide nanoparticles and 5‐bromo‐2‐deoxyuridine, and compared the migration pattern of NPCs in acute inflamed (n = 10) versus chronic demyelinated (n = 9) brains of mice induced with experimental allergic encephalomyelitis (EAE). Serial in vivo and ex‐vivo 3D magnetic resonance imaging revealed that NPCs migrated 2.5 ± 1.3 mm along the corpus callosum in acute EAE. In chronic EAE, cell migration was slightly reduced (2.3 ± 1.3 mm) and only occurred in the lateral side of transplantation. Surprisingly, in 6/10 acute EAE brains, NPCs were found to migrate in a radial pattern along RECA‐1⁺ cortical blood vessels, in a pattern hitherto only reported for migrating glioblastoma cells. This striking radial biodistribution pattern was not detected in either chronic EAE or disease‐free control brains. In both acute and chronic EAE brain, Iba1⁺ microglia/macrophage number was significantly higher in central nervous system regions containing migrating NPCs. The existence of differential NPC migration patterns is an important consideration for implementing future translational studies in multiple sclerosis patients with variable disease. Magn Reson Med, 2011. © 2011 Wiley‐Liss, Inc.     

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.

LacZ-transfected C17.2 neural stem cells (NSCs) were labeled with the superparamagnetic iron oxide formulation Feridex prior to ICV injection in shi/shi neonates. Feridex labeling did not alter cell differentiation in vitro and in vivo. Initially, MR images obtained at 11.7T correlated closely to NSC distribution as assessed with anti-dextran and anti-beta-galactosidase double-fluorescent immunostaining. However, at 6 days postgrafting there was already a pronounced mismatch between the hypointense MR signal and the histologically determined cell distribution, with a surprisingly sharp cutoff rather than a gradual decrease of signal. Positive in vivo BrdU labeling of NSCs showed that significant cell replication occurred post-transplantation, causing rapid dilution of Feridex particles between mother and daughter cells toward undetectable levels. Neural differentiation experiments demonstrated asymmetric cell division, explaining the observed sharp cutoff. At later time points (2 weeks), the mismatch further increased by the presence of non-cell-associated Feridex particles resulting from active excretion or cell death. These results are a first demonstration of the inability of MRI to track rapidly dividing and self-renewing, asymmetrically dividing SCs. Therefore, MR cell tracking should only be applied for nonproliferating cells or short-term monitoring of highly-proliferative cells, with mitotic symmetry or asymmetry being important for determining its applicability.     

肾功能衰竭大鼠超顺磁性氧化铁标记骨髓间充质干细胞肾脏移植MR活体示踪的研究

目的应用磁性氧化铁纳米粒子和多聚左旋赖氨酸（poly-L-lysine，PLL）的偶联物Fe2O3-PLL标记大鼠骨髓间充质干细胞（MSCs），MR活体示踪经肾动脉移植入肾功能衰竭（简称肾衰）大鼠肾脏的标记细胞。方法制备Fe2O3-PLL，分离、纯化并培养大鼠骨髓MSCs，Fe2O3-PLL标记细胞，普鲁士蓝染色显示细胞内铁。肌内注射甘油所致肾衰的大鼠分为2组，分别经左肾动脉移植入标记细胞（6只）和未标记细胞（5只），移植后即刻及第1、3、5、8天应用MRI对移植细胞进行活体示踪，并与肾脏组织切片普鲁士蓝染色和HE染色对照。结果MSCs的Fe2O3-PLL标记率近100％，普鲁士蓝染色显示蓝色铁颗粒位于MSCs胞质内。标记细胞移植后肾衰大鼠肾脏皮质区信号强度明显下降，T2＊WI信号改变最明显，而肾髓质及肾盂信号较细胞移植前无明显变化，信号改变随着时间的延长逐渐减轻一直持续到移植后第8天。组织学分析见绝大多数标记细胞分布于肾皮质肾小球内，与MRI信号改变区域基本一致。未标记细胞移植后未见肾脏信号改变。结论Fe2O3-PLL可以有效标记大鼠骨髓MSCs，临床应用型1．5T磁共振仪可对经肾动脉移植入肾衰大鼠肾脏的标记细胞进行初步活体示踪。     

脑梗死大鼠脑内移植超顺磁性氧化铁颗粒标记神经干细胞后的MR示踪研究

目的探讨超顺磁性氧化铁颗粒（SP10）标记的胎鼠神经干细胞（NSCs）在脑梗死模型大鼠脑内移植后，MR示踪观察的可行性。方法大鼠脑梗死模型24只，按随机数字表法分为3组：第1组大鼠同侧尾状核移植SP10和5-溴脱氧尿核苷（BrdU）双标记的NSCs；第2组对侧尾状核移植双标记的NSCs；第3组对侧尾状核移植未标记的NSCs。移植后1、3、5、7周后进行MR示踪观察，选择T2WI和梯度回波（GRE）序列，成像后相应时间点每组处死2只大鼠，取脑组织冰冻切片后进行普鲁士蓝染色及BrdU染色。结果移植后1周MRI显示：移植标记细胞组在注射点处可见类圆形低信号影，未标记细胞组注射点未见异常信号影；3周后，第1组梗死皮层下可见线状低信号影；移植5周后，第2组沿胼胝体走行可见扇形低信号影，尖端指向病灶。GRE序列显示标记细胞较清晰，而T2WI显示梗死病灶和大鼠脑正常结构较清晰。相应时间点相应部位普鲁士蓝染色及BrdU染色可见阳性细胞，与MRI结果相符。结论超顺磁性氧化铁颗粒和BrdU双标记的神经干细胞移植至大鼠脑内后可迁移到病灶区；MR成像能够在活体内连续示踪观察神经干细胞的迁移及分布情况。     

Volume correction for edema in single-volume proton MR spectroscopy of contrast-enhancing multiple sclerosis lesions.

The effect of edema on metabolic changes in contrast-enhancing multiple sclerosis lesions was studied by combining quantification of proton MR spectra with segmentation of the volume-of-interest, which was based on biexponential T(2) relaxation. All lesions showed a second component (s(long)) with a longer T(2) (185-450 ms), which was increased compared to healthy controls. Regression analysis indicated that s(long) replaces the short-T(2) component and total creatine. Since the water content was close to 100%, s(long) was used to correct for an increase in extracellular space. This compensated for the apparent loss of creatine and rendered cholines markedly increased, as observed in animals with experimental allergic encephalomyelitis. Total N-acetyl aspartate (NAA) concentration was inversely correlated with s(long) and between 34-70% of its average reduction was assigned to edema. Thus, NAA loss exceeded cellular loss. Assessment of varying degrees of edema may be especially beneficial for quantitative longitudinal studies.     

Imaging inflammation: direct visualization of perivascular cuffing in EAE by magnetic resonance microscopy

PURPOSE: To determine if the architectural features revealed by magnetic resonance microscopy (MRM) allow one to detect microscopic abnormalities associated with neuroinflammation in fixed brain sections from animals with experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). MATERIALS AND METHODS: Imaging was performed at the Center for In Vivo Microscopy (CIVM) using a 9.4-Tesla, 89-mm bore, superconducting magnet with actively shielded gradients capable of 850 mT/m. A number of MR contrasts and spatial resolutions were explored. RESULTS: The assessment of EAE brain showed that it is possible to visualize perivascular cuffing in vitro by MRM on three-dimensional T1 proton stains. CONCLUSION: Inflammatory cell infiltration is a prerequisite for the development of lesions in EAE and MS. Thus, the ability to directly detect individual perivascular cuffs of inflammation may provide a useful means of monitoring the time course of inflammatory events, as conventional histopathological scoring of perivascular cuffs is utilized, but in the absence of sectioning and staining.     

干细胞移植的磁性标记及磁共振成像活体示踪

干细胞移植的临床应用需要解决植入活体内干细胞在体内存活、迁移及分化的监测问题。通过对干细胞进行顺磁性标记,磁共振成像(MRI)能够在活体上显示标记的干细胞,并进行特异性地追踪及定位,是目前干细胞活体示踪极具前景的方法。干细胞进行磁性标记主要利用铁类或钆类对比剂,两者各有优缺点。利用铁类或钆类对比剂标记干细胞并进行MRI活体监测取得了成功。并在心脑缺血损伤的疾病模型中得到应用,但在干细胞磁性标记的载体选用及其标记率、标记的持久性、标记对细胞活力及遗传性状方面尚存在一定的问题。     

低浓度超顺磁性氧化铁标记兔骨髓间充质干细胞的体外磁共振成像研究

目的:以浓度为25μg Fe/ml的超顺磁性氧化铁纳米粒子(SPIO)体外标记兔骨髓间充质干细胞(BMSCs),并探讨1.5 T核磁共振仪成像的特征和成像所需最低标记细胞浓度,以及在标记后1 d、1周、2周、3周、4周的信号变化特征。方法:分离、纯化、培养兔BMSCs并以25μg Fe/ml的SPIO培养液浓度标记,对标记后不同时间的细胞行普鲁士蓝染色和台盼蓝拒染后显微镜观察,并进行MR成像,测量不同序列下不同浓度标记细胞管的信号强度,以确定扫描敏感序列及成像所需最低标记细胞浓度;再测量不同细胞浓度不同时相信号强度,来观察信号强度随时间变化的规律,并进行统计学分析。结果:浓度为25μg Fe/ml的超顺磁性氧化铁纳米粒子标记BMSCs的有效率接近100%,普鲁士蓝染色见细胞浆内有大小不等的蓝染铁颗粒,且在标记后4周内细胞仍具有活力,标记后的BMSCs在T2WI、尤其是GRE(T2*WI)序列信号明显降低;并且细胞浓度越高信号降低越明显,GRE序列MR成像的最低细胞浓度为5×104/ml。当标记细胞浓度为5×104/ml时,信号在T2*WI序列的降低2周后失去统计学意义;而在细胞浓度为5×105/ml时,标记3周后,信号在T2*WI序列的降低才失去统计学意义。结论:25μg/ml铁浓度标记干细胞不仅标记效率高,而且对细胞生长及增殖活力无明显影响,标记后MR信号改变与干细胞数目及标记时间相关。     

Quantitative imaging of magnetization transfer exchange and relaxation properties in vivo using MRI.

We describe a novel imaging technique that yields all of the observable properties of the binary spin-bath model for magnetization transfer (MT) and demonstrate this method for in vivo studies of the human head. Based on a new model of the steady-state behavior of the magnetization during a pulsed MT-weighted imaging sequence, this approach yields parametric images of the fractional size of the restricted pool, the magnetization exchange rate, the T(2) of the restricted pool, as well as the relaxation times in the free pool. Validated experimentally on agar gels and samples of uncooked beef, we demonstrate the method's application on two normal subjects and a patient with multiple sclerosis.     

In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction.

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 microg Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10-microl aliquots of 10(7) SPIO-loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. These findings support the ability of MRI to test the long-term therapeutic potential of embryonic stem cells in small animals in the setting of myocardial infarction.     

In vivo 4.0-T magnetic resonance investigation of spinal cord inflammation, demyelination, and axonal damage in chronic-progressive experimental allergic encephalomyelitis

PURPOSE: To image and dissect the lumbar spinal cord of guinea pigs with chronic-progressive experimental allergic encephalomyelitis (CP-EAE) and directly correlate the pathology to the magnetic resonance (MR) image data obtained at 4 T and determine if these MR contrasts can accurately differentiate a specific type of pathology from control tissue. MATERIALS AND METHODS: The amount of inflammation, demyelination, and axonal pathology were quantified in the whole cord cross sections. The signal intensities (SIs) for 228 individual regions of interest (ROIs) (normal-appearing white matter (NAWM) and tissue containing inflammation with or without demyelination) were measured directly from the corresponding area on the MR images. RESULTS: Conventional MR contrast SIs and magnetization transfer ratio (MTR) were related to the degree of demyelination and presence of inflammation. MTR and proton density-weighted (PDw) SIs were both moderately related to axonal density. The SIs for NAWM and in lesions containing both cellular infiltrates and demyelination in all conventional MR contrast images were also increased, whereas the MTR was decreased when compared to control tissue. CONCLUSION: The SIs from the conventional MR contrasts and MTR at 4 T were sensitive to the presence of disease within CP-EAE spinal cord, but were not specific to the underlying pathology.     

Magnetic resonance techniques for the in vivo assessment of multiple sclerosis pathology: consensus report of the white matter study group

On October 9-11, 2003, the third meeting of the White Matter Study Group of the International Society for Magnetic Resonance in Medicine was held in Venice, Italy. This article is the report of the meeting on how to use MRI in the diagnostic workup of multiple sclerosis (MS) and allied white matter disorders, and to define the nature and the extent of MS pathology in vivo. Both of these steps are central to the design of future treatment strategies aimed at limiting the functional consequences of the most disabling aspects of this disease.