Enhancement of regeneration with glia cell line–derived neurotrophic factor–transduced human amniotic fluid mesenchymal stem cells after sciatic nerve crush injury [RETRACTED]

Laboratory investigation

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Object

Human amniotic fluid–derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regeneration capacity. The present study evaluates whether the transplantation of glia cell line–derived neurotrophic factor (GDNF)–modified human AFMSCs may enhance regeneration of sciatic nerve after a crush injury.

Methods

Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. Either GDNF-modified human AFMSCs or human AFMSCs were embedded in Matrigel and delivered to the injured nerve. Motor function and electrophysiological studies were conducted after 1 and 4 weeks. Early or later nerve regeneration markers were used to evaluate nerve regeneration. The expression of GDNF in the transplanted human AFMSCs and GDNF-modified human AFMSCs was monitored at 7-day intervals.

Results

Human AFMSCs were successfully transfected with adenovirus, and a significant amount of GDNF was detected in human AFMSCs or the culture medium supernatant. Increases in the sciatic nerve function index, the compound muscle action potential ratio, conduction latency, and muscle weight were found in the groups treated with human AFMSCs or GDNF-modified human AFMSCs. Importantly, the GDNF-modified human AFMSCs induced the greatest improvement. Expression of markers of early nerve regeneration, such as increased expression of neurofilament and BrdU and reduced Schwann cell apoptosis, as well as late regeneration markers, consisting of reduced vacuole counts, increased expression of Luxol fast blue and S100 protein, paralleled the results of motor function. The expression of GDNF in GDNF-modified human AFMSCs was demonstrated up to 4 weeks; however, the expression decreased over time.

Conclusions

The GDNF-modified human AFMSCs appeared to promote nerve regeneration. The consecutive expression of GDNF was demonstrated in GDNF-modified human AFMSCs up to 4 weeks. These findings support a nerve regeneration scenario involving cell transplantation with additional neurotrophic factor secretion.

Abbreviations used in this paper: AFMSC = amniotic fluid–derived mesenchymal stem cell; BDNF = brain-derived neurotrophic factor; CMAP = compound muscle action potential; ELISA = enzyme linked immunosorbent assay; GDNF = glia cell line–derived neurotrophic factor; GFP = green fluorescent protein; MOI = multiplicity of infection; NGF = nerve growth factor; NT-3 = neurotrophin-3; SFI = sciatic nerve function index.

Article Information

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

Please include this information when citing this paper: published online October 9, 2009; DOI: 10.3171/2009.8.JNS09850.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    In vitro assay of GDNF in GDNF-modified AFMSCs. The AFMSCs were transfected either with rAd-GFP or rAd-GDNF for 48 hours. A: Photomicrographs in light phase (upper row) and corresponding images (bottom row) showing expression of GFP in AFMSCs after rAd-GFP infection. B: Western blot analyses of GDNF in Ad-GFP AFMSCs and Ad-GDNF AFMSCs. C: Graph showing results of ELISA analysis of supernatant with respect to Ad-GFP AFMSCs and Ad-GDNF AFMSCs.

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    Evaluation of SFI, electrophysiological function, and muscle weight. The left sciatic nerve was crushed; SFI, CMAP, conduction latency, and muscle weight were investigated in 6 animals per group. Upper: Graph showing representative scores of SFI. Lower: Ratio of CMAP, conduction latency, and muscle weight. AFS = AFMSC Group; AFS-G = GDNF-AFMSC Group (rats treated with GDNF-modified AFMSCs); Crush = Crush Group; Crush + M = Crush + Matrigel Group; LT = left; Rt = right. *** p < 0.001; ### p < 0.001 relative to AFMSCs.

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    Expression of neurofilament and results of TUNEL and BrdU assay in crushed nerve 7 days after injury. A: Representative example of results of staining for neurofilament, TUNEL, and BrdU assay in crushed nerve. Bar = 50 μm. Insets: Merging of TUNEL and BrdU staining with S100 staining (red). B and C: Quantitative analyses of expression of neurofilament levels (B), TUNEL results (C), and BrdU staining in 6 animals. *** p < 0.001; ** p < 0.01; ### p < 0.001 relative to AFMSCs.

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    Vacuole counts and Luxol fast blue and S100 staining in 6 animals 4 weeks postinjury. A: Representative photomicrographs illustrating vacuoles, Luxol fast blue staining, and S100 staining in each group. Bar = 50 μm. B: Quantitative analysis of vacuole counts. C: Quantitative analyses of Luxol fast blue and S100 staining. *** p < 0.001; ** p < 0.01; ### p < 0.001; ## p < 0.01 relative to AFMSCs.

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    Expression of GDNF in GDNF-modified AFMSCs. Left sciatic nerve specimens obtained at 7-day intervals were subjected to immunohistochemical analysis with antibodies to GDNF and the GDNF receptor. A: Photomicrographs showing decreased expression of GDNF (green) colocalized with GDNF-modified AFMSCs (red) at different time points, with AFMSCs (AFS) as a control. Bar = 50 μm. B: Representative photomicrographs showing expression of GDNF receptor in the distal end of crushed nerve from animals in the 4 different groups 7 days after injury. Bar = 50 μm. C: Quantitative analyses of GDNF and CM Dil counts. D: Quantitative analysis of GDNF receptor expression in the distal end of the crushed nerve. *** p < 0.001; ** p < 0.01; ### p < 0.001 relative to the AFMSC Group.

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