Recruitment by SDF-1α of CD34-positive cells involved in sciatic nerve regeneration

Laboratory investigation

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Object

Increased integration of CD34+ cells in injured nerve significantly promotes nerve regeneration, but this effect can be counteracted by limited migration and short survival of CD34+ cells. SDF-1α and its receptor mediate the recruitment of CD34+ cells involved in the repair mechanism of several neurological diseases. In this study, the authors investigate the potentiation of CD34+ cell recruitment triggered by SDF-1α and the involvement of CD34+ cells in peripheral nerve regeneration.

Methods

Peripheral nerve injury was induced in 147 Sprague-Dawley rats by crushing the left sciatic nerve with a vessel clamp. The animals were allocated to 3 groups: Group 1, crush injury (controls); Group 2, crush injury and local application of SDF-1α recombinant proteins; and Group 3, crush injury and local application of SDF-1α antibody. Electrophysiological studies and assessment of regeneration markers were conducted at 4 weeks after injury; neurobehavioral studies were conducted at 1, 2, 3, and 4 weeks after injury. The expression of SDF-1α, accumulation of CD34+ cells, immune cells, and angiogenesis factors in injured nerves were evaluated at 1, 3, 7, 10, 14, 21, and 28 days after injury.

Results

Application of SDF-1α increased the migration of CD34+ cells in vitro, and this effect was dose dependent. Crush injury induced the expression of SDF-1α, with a peak of 10–14 days postinjury, and this increased expression of SDF-1α paralleled the deposition of CD34+ cells, expression of VEGF, and expression of neurofilament. These effects were further enhanced by the administration of SDF-1α recombinant protein and abolished by administration of SDF-1α antibody. Furthermore, these effects were consistent with improvement in measures of neurological function such as sciatic function index, electrophysiological parameters, muscle weight, and myelination of regenerative nerve.

Conclusions

Expression of SDF-1α facilitates recruitment of CD34+ cells in peripheral nerve injury. The increased deposition of CD34+ cells paralleled significant expression of angiogenesis factors and was consistent with improvement of neurological function. Utilization of SDF-1α for enhancing the recruitment of CD34+ cells involved in peripheral nerve regeneration may be considered as an alternative treatment strategy in peripheral nerve disorders.

Abbreviations used in this paper: CMAP = compound muscle action potential; ECM = extracellular matrix; G-CSF = granulocyte colony-stimulating factor; HPC = hematopoietic progenitor cell; RI = regularity index; 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-Kang Road, Section 3, Taichung 407, Taiwan. email: hcpan2003@yahoo.com.tw.

Please include this information when citing this paper: published online August 19, 2011; DOI: 10.3171/2011.3.JNS101582.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Migration of HPCs treated with SDF-1α. A: Photomicrographs showing transwell migration assay. Original magnification × 200. Bar = 50 μm. B: Quantitative analysis of cell numbers of field (HPCs per 0.01 mm2). ***p < 0.001 (relative to SDF-1α 5 μg/ml); ###p < 0.01 (relative to SDF-1α 10 μg/ml).

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    Expression of SDF-1α and associated protein after sciatic nerve crush injury. A: Expression of SDF-1α, CXCR-4, CD68, and CD34 at different time intervals; β-actin was used for calibration. B: Results of quantitative analysis at 1, 3, 7, 10, and 14 days after crush injury. IAU = intensity arbitrary units; L = left sciatic nerve (crushed with vessel clamps); R = right sciatic nerve (control). *p < 0.05, **p < 0.01, ***p < 0.001 (vs Day 1).

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    Expression of SDF-1α profiles treated with either neutralized antibodies or SDF-1α. A: Expression of SDF-1α in right and left sciatic nerves treated with neutralized antibodies or SDF-1α recombinant proteins at different time intervals. B: Quantitative analysis of SDF-1α based on treatment groups and time intervals. C = crush injury (left sciatic nerve); N = neutralized antibody; R = right sciatic nerve as a control. **p < 0.01; ***p < 0.001 (vs crush injury group [untreated controls]).

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    Expression of angiogenesis factors. A: Expression of CD68, CD34, and VEGF based on different treatment modalities. B–D: Quantitative analysis of CD68, CD34, and VEGF. **p < 0.01; ***p < 0.001 (vs controls).

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    Results of neurobehavioral and electrophysiological studies in rats with untreated crush injury (crush, solid line), crush injury treated with neutralized SDF-1α antibody (NA, small-dash line), and crush injury treated with SDF-1α recombinant protein (SDF-1, largedash line). A and B: Mean SFI values and ankle angles (in degrees) at different time points for the 3 groups. C: Quantitative analysis of the ratios of muscle weight (MW), CMAP, and conduction latency in left (injured) and right (control) legs. Mean values from 6 animals per group. Lt/Rt = left/right. *p < 0.05; **p < 0.01 (vs controls).

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    CatWalk analysis based on different treatment modalities and time points. A: Ratio of mean intensity values (left [injured side]/right [uninjured]). B: Paw print area ratio (left/right). C: Stance duration ratio (left/right). D: Swing duration ratio (left/right). Values are group means; 6 rats per group. L/R = left/right. *p < 0.05, **p < 0.01 (vs controls).

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    Histological findings in sciatic nerve injury. Left sciatic nerves were crushed and treated with either neutralized antibodies or SDF-1α recombinant protein or left untreated. The nerves were retrieved for analysis 28 days after injury. A: Photomicrographs showing expression of S100 and relative numbers of vacuoles in the 3 treatment groups. B and C: Quantitative analysis of S100 expression (B) and vacuole counts (C). *p < 0.05, **p < 0.01, ***p < 0.001 (vs controls).

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    Expression of CD68, CD34, and neurofilament (NF) based on different treatment modalities and time intervals. A: Photomicrographs showing CD68, CD34, and neurofilament expression at different time intervals. Bar = 50 μm. B–D: Results of quantitative analysis of CD68, CD34, and neurofilament expression. *p < 0.05, **p < 0.01, ***p < 0.001 (vs controls).

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