The recovery of blood-nerve barrier in crush nerve injury--a quantitative analysis utilizing immunohistochemistry.
ABSTRACT The purpose of this study is to reveal whether the application of immunohistochemical examinations to the peripheral nervous system (PNS) can be a reliable method for the quantitative analysis of the blood-nerve barrier (BNB) and the relationship between restoration of BNB and nerve regeneration. Sciatic nerves in rats were examined after nerve crush. Immunohistochemical staining with anti-rat endothelial cell antigen-1 (anti-RECA-1) that recognizes endothelial cells and anti-endothelial barrier antigen (anti-EBA) for the detection of barrier-type endothelial cells were used. Neurofilament for staining axons was also performed. A quantitative analysis of the BNB was assessed using the ratio of EBA positive cells and RECA-1 positive cells. The ratio of EBA/RECA-1 decreased significantly 3 days postoperatively and reached its lowest level at day 7 in the segment 5 mm proximal and the entire distal stump. The ratio gradually recovered from the proximal and the regeneration of axons started a week earlier than BNB. The ratio of EBA/RECA-1 applied to the PNS can be a reliable method for the quantitative analysis of BNB. In crush injuries, the breakdown of BNB occurred simultaneously in the segment 5 mm proximal and the entire distal stump; restoration began from the proximal to distal and followed a week later to nerve regeneration.
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ABSTRACT: 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. 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. 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. 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.Journal of Neurosurgery 10/2009; 112(4):868-79. · 3.15 Impact Factor
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ABSTRACT: Object Mobilization of hematopoietic progenitor cells (HPCs) from bone marrow involved in the process of peripheral nerve regeneration occurs mostly through deposits of CD34(+) cells. Treadmill exercise, with either differing intensity or duration, has been shown to increase axon regeneration and sprouting, but the effect of mobilization of HPCs on peripheral nerve regeneration due to treadmill exercise has not yet been elucidated. Methods Peripheral nerve injury was induced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were categorized into 2 groups: those with and without treadmill exercise (20 m/min for 60 minutes per day for 7 days). Cytospin and flow cytometry were used to determine bone marrow progenitor cell density and distribution. Neurobehavioral analysis, electrophysiological study, and regeneration marker expression were investigated at 1 and 3 weeks after exercise. The accumulation of HPCs, immune cells, and angiogenesis factors in injured nerves was determined. A separate chimeric mice study was conducted to assess CD34(+) cell distribution according to treadmill exercise group. Results Treadmill exercise significantly promoted nerve regeneration. Increased Schwann cell proliferation, increased neurofilament expression, and decreased Schwann cell apoptosis were observed 7 days after treadmill exercise. Elevated expression of S100 and Luxol fast blue, as well as decreased numbers of vacuoles, were identified in the crushed nerve 3 weeks after treadmill exercise. Significantly increased numbers of mononuclear cells, particularly CD34(+) cells, were induced in bone marrow after treadmill exercise. The deposition of CD34(+) cells was abolished by bone marrow irradiation. In addition, deposits of CD34(+) cells in crushed nerves paralleled the elevated expressions of von Willebrand factor, isolectin B4, and vascular endothelial growth factor. Conclusions Bone marrow HPCs, especially CD34(+) cells, were able to be mobilized by low-intensity treadmill exercise, and this effect paralleled the significant expression of angiogenesis factors. Treadmill exercise stimulation of HPC mobilization during peripheral nerve regeneration could be used as a therapy in human beings.Journal of Neurosurgery 11/2012; · 3.15 Impact Factor
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ABSTRACT: 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. 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. 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. 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.Journal of Neurosurgery 08/2011; 116(2):432-44. · 3.15 Impact Factor