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. 2017 Aug 1;8(35):58985-58999.
doi: 10.18632/oncotarget.19775. eCollection 2017 Aug 29.

Magnetic resonance imaging tracking and assessing repair function of the bone marrow mesenchymal stem cells transplantation in a rat model of spinal cord injury

Hongwu Zhang #  1   2 , Liqin Wang #  3 , Shihong Wen  1 , Qingfeng Xiang  4 , Xianhong Xiang  3 , Caixia Xu  1 , Yong Wan  5 , Jingnan Wang  1 , Bin Li  1 , Yiqian Wan  6 , Zhiyun Yang  3 , David Y B Deng  1   7
Affiliations

Magnetic resonance imaging tracking and assessing repair function of the bone marrow mesenchymal stem cells transplantation in a rat model of spinal cord injury

Hongwu Zhang et al. Oncotarget. .

Abstract

The transplantation of bone marrow mesenchymal stem cells (BMSCs) to repair spinal cord injury (SCI) has become a promising therapy. However, there is still a lack of visual evidence directly implicating the transplanted cells as the source of the improvement of spinal cord function. In this study, BMSCs were labeled with NF-200 promoter and lipase-activated gadolinium-containing nanoparticles (Gd-DTPA-FA). Double labeled BMSCs were implanted into spinal cord transaction injury in rat models in situ, the function recovery was evaluated on 1st, 7th, 14th, 28 th days by MRI, Diffusion Tensor Imaing, CT imaging and post-processing, and histological observations. BBB scores were used for assessing function recovery. After transplantation of BMSCs, the hypersignal emerged in spinal cord in T1WI starting at day 7 that was focused at the injection site, which then increased and extended until day 14. Subsequently, the increased signal intensity area rapidly spread from the injection site to entire injured segment lasting four weeks. The diffusion tensor tractography and histological analysis both showed the nerve fibre from dividing to connecting partly. Immunofluorescence showed higher expression of NF-200 in Repaired group than Injury group. Electron microscopy showed detachment and loose of myelin lamellar getting better in Repaired group compared with the Injury group. BBB scores in Repaired group were significantly higher than those of injury animals. Our study suggests that the migration and distribution of Gd-DTPA-FA labeled BMSCs can be tracked using MRI. Transplantation of BMSCs represents a promising potential strategy for the repair of SCI.

Keywords: Gd-DTPA-FA; bone marrow mesenchymal stem cells; endothelial lipase; magnetic resonance imaging; spinal cord injury.

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Conflict of interest statement

CONFLICTS OF INTEREST All the authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Immunophenotypic analysis of BMSCs and the construction of NF-200 promoter of lentivirus plasmid and identification
(A) Flow cytometry showed negative expression of CD45 and CD11b/c while positive expression of CD29 and CD90. (B) Confocal microscopy images to confirm the neurogenic differentiation of BMSCs by the neurocyte-specific markers NSE and NF-200. Hoechst33342 a blue tint to the nucleus, NF-200 dyed red, and NSE dyed green. (C) Scheme of plasmid. (D) Agarose gel electrophoresis of EL (endothelial lipase) DNA. (E) PRRL. sin. CMV. EL-eGFP digested with DraI and NotI enzymes1, 3, 5: DNA extracted from three stochastic monoclonal colonies of PRRL. sin. CMV. EL-eGFP; 2, 4, 6: Double digestion results of 1, 3, 5 with SpeI and EcoRI. (F) Sequencing of the recombined PRRL. sin. CMV. EL-eGFP verctor. a: The sequenceing after Pcmv; b: The sequenceing before IRES. (G) Production of recombined lentivirus and infection with HepG2 cells. a: Fluorescent image of 293T cells transfected with PRRL. sin. CMV. EL-eGFP verctor; b: Overlap image of 293T cells transfected with PRRL. sin. CMV. EL-eGFP verctor; c: Bright image of 293T cells transfected with PRRL. sin. CMV. EL-eGFP verctor; d: Fluorescent image of HepG2 cells transfected with PRRL. sin. CMV. EL-eGFP lentivirus; e: Overlap image of HepG2 cells infected with PRRL. sin. CMV. EL-eGFP lentivirus; f: Bright image of HepG2 cells transfected with PRRL. sin. CMV. EL-eGFP lentivirus. (H) Expressing of EL mRNA in HepG2 cells. a: untransfected HepG2 cells; b: transfected HepG2 cells with PRRL. sin. CMV. eGFP lentivirus; c: transfected HepG2 cells with PRRL. sin. CMV. EL-eGFP lentivirus; EL: endothelial lipase. (I) Expressing of EL protein in HepG2 cells. a. untransfected HepG2 cells; b. transfected HepG2 cells with PRRL. sin. CMV. eGFP lentivirus; c. infected HepG2 cells with PRRL. sin. CMV. EL-eGFP lentivirus.
Figure 2
Figure 2. MRI imaging of Normal group, Injury group and Repaired group in the rat SCI model at 1 d, 7 d, 14 d and 28 d
In Normal group, the rat spinal cord was continuous and uninterrupted. In Injury group, there was no observable increase in signal intensity of the spinal cord in T1WI from 1st day to 28th day. In contrast, hypersignal emerged in spinal cords of the Repaired group of rats in T1WI beginning on the 7th day, which was further observed to be increased and extended until the 14th day statu-post injection. On day 28, the area of hypersignal in spinal cord was larger than before. Arrows indicated the segmental SCI.
Figure 3
Figure 3. Nissl's staining and electron microscopy of rat spinal cord tissue
(A) Nissl's staining results showed that the numbers of neurons decreased gradually from the 1st day to 28th day in Injury group, and the damage degree increased gradually. In Repaired group, corresponding BMSCs transplantation could promote the survival of neurons, reduce the apoptosis of SCI (magnification: 100×; insert magnification: 400 ×). (B) The electron microscopy scans. The Injury group and Repaired group showed many demyelinating axons. In Injury group, the degree of pathological changes of ultrastructure over time decreased, and the ultrastructure of BMSCs transplantation group was obviously improved.
Figure 4
Figure 4. Diffusion tensor tractography (DTT) figures, H.E staining of rat spinal cord tissue and BBB locomotor function score
(A) The DTT. In Injury group, the spinal cord fibers continuity were interrupted from the 1st day to 28th day, and end fibers arranged disorderly, colors were uneven; In Repaired group, the nerve fibers from the 1st day to 28th day, some fibers connected to the other end. The color was almost consistent, but the arrangement of neurofibra was still disorder. (B) a. In Normal group, visible normal spinal structure; b. In Injury 1d group, spinal cord was completely broken; c. In Repaired 28 d group, continuous spinal nerve fibers appeared in SCI site. (C) BBB locomotor function score. BBB scores increased gradually from 2～8 weeks in BMSCs repaired group. After transplant 8 weeks, BBB scores (score > 8)were significantly higher than Injury group(score < 4), **P < 0.01.
Figure 5
Figure 5. Immunofluorescence of spinal cord tissue
NF-200 antibody (red), dyeing Hoechst33342 nucleus (blue). It showed that the pathological changes in Injury group decreased with time obviously in longitudinal section. The NF-200 positive cells expressing quantity increased significantly in Repaired group compared to Injury group (n = 5). Bar: 100 μm.
Figure 6
Figure 6. Schematic illustration of BMSCs transplantation differentiation, migration and distribution diagram
(A) Normal group, Injury group and Repaired group rats MRI signal intensity relatively statistical histogram. *P < 0.05 verus Normal, #P < 0.05 verus Injury. (B) The T1WI signal intensity distribution in Repaired group in different time (1, 7, 14, 28 days). The T1WI signal strength increased with the extension of time, at 14th day peaked, and then began to decline. The area of high signal intensity increased with the extension of time, and the biggest area of high signal intensity appeared at 28th day. The weighted total signal strength was on the rise. (C) Schematic illustration of BMSCs transplantation differentiation, migration and distribution diagram. In the 7th day, NF-200 positive cells were observed near the injection point, suggesting that the BMSCs had differentiated into neurons and were mainly concentrated at the site of SCI. The number of NF-200 positive cells reached a maximum at 14th day, and then gradually distributed along the segmental injury to 28th day, NF-200 positive cells distributed to both ends of the injury of spinal cord.
Cover Figure 1
Cover Figure 1. Schematic illustration for the paper's design
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