Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid–derived mesenchymal stem cells regulated by stromal cell–derived factor-1α in a sciatic nerve injury model

Laboratory investigation

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Object

Human amniotic fluid–derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration. The expression of stromal cell–derived factor-1α (SDF-1α) in the injured nerve exerts a trophic effect by recruiting progenitor cells that promote nerve regeneration. In this study, the authors investigated the feasibility of intravenous administration of AFMSCs according to SDF-1α expression time profiles to facilitate neural regeneration in a sciatic nerve crush injury model.

Methods

Peripheral nerve injury was induced in 63 Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into 1 of 3 groups: Group I, crush injury as the control; Group II, crush injury and intravenous administration of AFMSCs (5 × 106 cells for 3 days) immediately after injury (early administration); and Group III, crush injury and intravenous administration of AFMSCs (5 × 106 cells for 3 days) 7 days after injury (late administration). Evaluation of neurobehavior, electrophysiological study, and assessment of regeneration markers were conducted every week after injury. The expression of SDF-1α and neurotrophic factors and the distribution of AFMSCs in various time profiles were also assessed.

Results

Stromal cell–derived factor-1α increased the migration and wound healing of AFMSCs in vitro, and the migration ability was dose dependent. Crush injury induced the expression of SDF-1α at a peak of 10–14 days either in nerve or muscle, and this increased expression paralleled the expression of its receptor, chemokine receptor type-4 (CXCR-4). Most AFMSCs were distributed to the lung during early or late administration. Significant deposition of AFMSCs in nerve and muscle only occurred in the late administration group. Significantly enhanced neurobehavior, electrophysiological function, nerve myelination, and expression of neurotrophic factors and acetylcholine receptor were demonstrated in the late administration group.

Conclusions

Amniotic fluid–derived mesenchymal stem cells can be recruited by expression of SDF-1α in muscle and nerve after nerve crush injury. The increased deposition of AFMSCs paralleled the expression profiles of SDF-1α and its receptor CXCR-4 in either muscle or nerve. Administration of AFMSCs led to improvements in neurobehavior and expression of regeneration markers. Intravenous administration of AFMSCs may be a promising alternative treatment strategy in peripheral nerve disorder.

Abbreviations used in this paper:AFMSC = amniotic fluid–derived MSC; BDNF = brain-derived neurotrophic factor; CMAP = compound muscle action potential; CNTF = ciliary neurotrophic factor; CXCR-4 = chemokine receptor type-4; DMSO = dimethyl sulfoxide; FBS = fetal bovine serum; GDNF = glia-derived neurotrophic factor; MSC = mesenchymal stem cell; NT-3 = neurotrophin-3; SDF-1α = stromal cell–derived factor-1α; SFI = sciatic function index.

Article Information

Address correspondence to: Hung-Chuan Pan, M.D., Ph.D., Department of Neurosurgery, Taichung Veterans General Hospital, No. 160, Taichung Port Road, Sec. 3, Taichung 407, Taiwan. email: hcpan2003@yahoo.com.tw.

Please include this information when citing this paper: published online April 13, 2012; DOI: 10.3171/2012.2.JNS111360.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Images of the migration ability of AFMSCs treated with SDF-1α. A: Photomicrographs showing the migration ability of AFMSCs over time (hours) in the wound healing study. Original magnification × 400. B: Representative example of Giemsa staining in the transwell migration assay after 12 hours. Bar = 100 μm. C: Quantitative analysis of Giemsa staining in the transwell migration assay presented as number of cells per field. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the 0 ng/ml SDF-1α group. N = 3 samples tested.

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    Expression of SDF-1α and CXCR-4 in AFMSCs, crushed nerve, and muscle. Retrieved nerve and muscle tissues were obtained for Western blot analysis at different time points. A: Immunohistochemical staining of SDF-1α and CXCR-4 in AFMSCs. Bar = 100 μm. B: Western blot analysis of SDF-1α and CXCR-4 in nerve and muscle tissues at different time intervals. C and D: Quantitative analysis of SDF-1α and CXCR-4 in nerve (C) and muscle tissues (D) at different time intervals. *p < 0.05, **p < 0.01 as compared with values after 3 days. IAU = intensity arbitrary units. N = 3 samples tested.

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    Ultraviolet microscopy (350 nm) shows distribution of AFMSCs in the lung (A–D), spleen (E–H), nerve (I–L), and muscle (M–P) in early or late intravenous administration of AFMSCs. Bar = 50 μm; N = 3 samples tested.

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    Graphs showing results of the neurobehavioral and electrophysiological studies. Representative SFI (A) and quantitative analysis of the ratio of left/right muscle weight (MW), CMAP, and conduction latency (B). *p < 0.05, **p < 0.01. N = 6 animals per group.

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    Graphs of automated quantitative gait analysis based on different treatment modalities and time points. A: Ratio of mean intensity (left/right). B: Printed area ratio (left/right). C: Stance duration ratio (left/right). D: Swing duration ratio (left/right). *p < 0.05, **p < 0.01. N = 6 animals per group.

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    Images showing the size and number of axons, as well as acetylcholine receptor density 4 weeks after injury. A: Representative photomicrograph showing morphology and number of axons in the distal end of the crushed nerve. AFS = AFMSC. B: Representative photomicrograph showing morphology of the acetylcholine (Ach) receptor in the gastrocnemius muscle. Bar = 100 μm. C: Quantitative analysis of various sizes of axons. *p < 0.05, **p < 0.01, ##p < 0.01 (AFS 7 days compared with AFS 0 days). D: Quantitative analysis of the density of the acetylcholine receptor in the gastrocnemius muscle. #p < 0.05 (AFS 7 days compared with AFS 0 days), ***p < 0.001. N = 3 samples tested.

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    Distribution of AFMSCs in nerve and muscle in late administration. Photomicrographs show double immunohistochemical staining in epineuria (A) and muscle (B) using Hoechst 33342 for BDNF, CNTF, NT-3, and S100. Bar = 100 μm. N = 3 samples tested.

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