Canbin Zheng

Effect of platelet-rich plasma (PRP) concentration on proliferation, neurotrophic function and migration of Schwann cells in vitro.

Platelet‐rich plasma (PRP) contains various growth factors and appears to have the potential to promote peripheral nerve regeneration, but evidence is lacking regarding its biological effect on Schwann cells (SCs). The present study was designed to investigate the effect of PRP concentration on SCs in order to determine the plausibility of using this plasma‐derived therapy for peripheral nerve injury. PRP was obtained from rats by double‐step centrifugation and was characterized by determining platelet numbers and growth factor concentrations. Primary cultures of rat SCs were exposed to various concentrations of PRP (40%, 20%, 10%, 5% and 2.5%). Cell proliferation assays and flow cytometry were performed to study to assess SC proliferation. Quantitative real‐time PCR and ELISA analysis were performed to determine the ability of PRP to induce SCs to produce nerve growth factor (NGF) and glial cell line‐derived neurotrophic factor (GDNF). Microchemotaxis assay was used to analyse the cell migration capacity. The results obtained indicated that the platelet concentration and growth factors in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 2.5–20% PRP significantly stimulated SC proliferation and migration compared to untreated controls in a dose‐dependent manner. In addition, the expression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by high PRP concentrations (40%). In conclusion, the appropriate concentration of PRP had the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors and significantly increased migration of SCs dose‐dependently. Copyright

Factors predicting sensory and motor recovery after the repair of upper limb peripheral nerve injuries

OBJECTIVE: To investigate the factors associated with sensory and motor recovery after the repair of upper limb peripheral nerve injuries. DATA SOURCES: The online PubMed database was searched for English articles describing outcomes after the repair of median, ulnar, radial, and digital nerve injuries in humans with a publication date between 1 January 1990 and 16 February 2011. STUDY SELECTION: The following types of article were selected: (1) clinical trials describing the repair of median, ulnar, radial, and digital nerve injuries published in English; and (2) studies that reported sufficient patient information, including age, mechanism of injury, nerve injured, injury location, defect length, repair time, repair method, and repair materials. SPSS 13.0 software was used to perform univariate and multivariate logistic regression analyses and to investigate the patient and intervention factors associated with outcomes. MAIN OUTCOME MEASURES: Sensory function was assessed using the Mackinnon-Dellon scale and motor function was assessed using the manual muscle test. Satisfactory motor recovery was defined as grade M4 or M5, and satisfactory sensory recovery was defined as grade S3+ or S4. RESULTS: Seventy-one articles were included in this study. Univariate and multivariate logistic regression analyses showed that repair time, repair materials, and nerve injured were independent predictors of outcome after the repair of nerve injuries (P < 0.05), and that the nerve injured was the main factor affecting the rate of good to excellent recovery. CONCLUSION: Predictors of outcome after the repair of peripheral nerve injuries include age, gender, repair time, repair materials, nerve injured, defect length, and duration of follow-up.

Etifoxine provides benefits in nerve repair with acellular nerve grafts

Introduction: Acellular nerve grafts are good candidates for nerve repair, but the clinical outcome of grafting is not always satisfactory. We investigated whether etifoxine could enhance nerve regeneration. Methods: Seventy‐two Sprague‐Dawley rats were divided into 3 groups: (1) autograft; (2) acellular nerve graft; and (3) acellular nerve graft plus etifoxine. Histological and electrophysiological examinations were performed to evaluate the efficacy of nerve regeneration. Walking‐track analysis was used to examine functional recovery. Quantitative polymerase chain reaction was used to evaluate changes in mRNA level. Results: Etifoxine: (i) increased expression of neurofilaments in regenerated axons; (ii) improved sciatic nerve regeneration measured by histological examination; (iii) increased nerve conduction velocity; (iv) improved walking behavior as measured by footprint analysis; and (v) boosted expression of neurotrophins. Conclusions: These results show that etifoxine can enhance peripheral nerve regeneration across large nerve gaps repaired by acellular nerve grafts by increasing expression of neurotrophins. Muscle Nerve 50:235–243, 2014

Etifoxine promotes glial‑derived neurotrophic factor‑induced neurite outgrowth in PC12 cells

Nerve regeneration and functional recovery are major issues following nerve tissue damage. Etifoxine is currently under investigation as a therapeutic strategy for promoting neuroprotection, accelerating axonal regeneration and modulating inflammation. In the present study, a well‑defined PC12 cell model was used to explore the underlying mechanism of etifoxine‑stimulated neurite outgrowth. Etifoxine was found to promote glial‑derived growth factor (GDNF)‑induced neurite outgrowth in PC12 cells. Average axon length increased from 50.29±9.73 to 22.46±5.62 µm with the use of etifoxine. However, blockage of GDNF downstream signaling was found to lead to the loss of this phenomenon. The average axon length of the etifoxine group reduces to a normal level after the blockage of the GDNF family receptor α1 (GFRα1) and receptor tyrosine kinase (RETS) receptors (27.46±3.59 vs. 22.46±5.62 µm and 25.31±3.68 µm vs. 22.46±5.62 µm, respectively, p>0.05). In addition, etifoxine markedly increased GDNF mRNA and protein expression (1.55‑ and 1.36-fold, respectively). However, blockage was not found to downregulate GDNF expression. The results of the current study demonstrated that etifoxine stimulated neurite outgrowth via GDNF, indicating that GDNF represents a key molecule in etifoxine‑stimulated neurite outgrowth in PC12 cells.

A novel mutation outside homeodomain of HOXD13 causes synpolydactyly in a Chinese family

INTRODUCTION Human synpolydactyly (SPD), belonging to syndactyly (SD) II, is caused by mutations in homeobox d13 (HOXD13). Here, we describe the study of a two-generation Chinese family with a variant form of synpolydactyly. MATERIALS AND METHODS The sequence of the HOXD13 gene was analyzed. Luciferase assays were conducted to determine whether the mutation affected the function of the HOXD13 protein. RESULTS We identified a novel c.659G>C (p.Gly220Ala) mutation outside the HOXD13 homeodomain responsible for the disease in this family. This mutation was not found in any of the unaffected family members and healthy control. Luciferase assays demonstrated that this mutation affected the transcriptional activation ability of HOXD13 (only approximately 84.7% of wild type, p<0.05). CONCLUSION Phenotypes displayed by individuals carrying the novel mutation present additional features, such as the fifth finger clinodactyly, which is not always associated with canonical SPD. This finding enhances our understanding about the phenotypic spectrum associated with HOXD13 mutations and advances our understanding of human limb development.

Analysis of human acellular nerve allograft reconstruction of 64 injured nerves in the hand and upper extremity: a 3 year follow-up study.

Human acellular nerve allografts have been increasingly applied in clinical practice. This study was undertaken to investigate the functional outcomes of nerve allograft reconstruction for nerve defects in the upper extremity. A total of 64 patients from 13 hospitals were available for this follow‐up study after nerve repair using human acellular nerve allografts. Sensory and motor recovery was examined according to the international standards for motor and sensory nerve recovery. Subgroup analysis and logistic regression analysis were conducted to identify the relationship between the known factors and the outcomes of nerve repair. Mean follow‐up time was 355 ± 158 (35–819) days; mean age was 35 ± 11 (14–68) years; average nerve gap length was 27 ± 13 (10–60) mm; no signs of infection, tissue rejection or extrusion were observed among the patients; 48/64 (75%) repaired nerves experienced meaningful recovery. Univariate analysis showed that site and gap length significantly influenced prognosis after nerve repair using nerve grafts. Delay had a marginally significant relationship with the outcome. A multivariate logistic regression model revealed that gap length was an independent predictor of nerve repair using human acellular nerve allografts. The results indicated that the human acellular nerve allograft facilitated safe and effective nerve reconstruction for nerve gaps 10–60 mm in length in the hand and upper extremity. Factors such as site and gap length had a statistically significant influence on the outcomes of nerve allograft reconstruction. Gap length was an independent predictor of nerve repair using human acellular nerve allografts. Copyright

Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury: Miconazole in Nerve Regeneration

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Methods: Fifty Sprague‐Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit‐8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57: 821–828, 2018

Iodine and freeze-drying enhanced high-resolution MicroCT imaging for reconstructing 3D intraneural topography of human peripheral nerve fascicles

BACKGROUND The precise annotation and accurate identification of the topography of fascicles to the end organs are prerequisites for studying human peripheral nerves. NEW METHOD In this study, we present a feasible imaging method that acquires 3D high-resolution (HR) topography of peripheral nerve fascicles using an iodine and freeze-drying (IFD) micro-computed tomography (microCT) method to greatly increase the contrast of fascicle images. RESULTS The enhanced microCT imaging method can facilitate the reconstruction of high-contrast HR fascicle images, fascicle segmentation and extraction, feature analysis, and the tracing of fascicle topography to end organs, which define fascicle functions. COMPARISON WITH EXISTING METHODS The complex intraneural aggregation and distribution of fascicles is typically assessed using histological techniques or MR imaging to acquire coarse axial three-dimensional (3D) maps. However, the disadvantages of histological techniques (static, axial manual registration, and data instability) and MR imaging (low-resolution) limit these applications in reconstructing the topography of nerve fascicles. CONCLUSIONS Thus, enhanced microCT is a new technique for acquiring 3D intraneural topography of the human peripheral nerve fascicles both to improve our understanding of neurobiological principles and to guide accurate repair in the clinic. Additionally, 3D microstructure data can be used as a biofabrication model, which in turn can be used to fabricate scaffolds to repair long nerve gaps.

The vascularization pattern of acellular nerve allografts after nerve repair in Sprague-Dawley rats

Abstract Objective: We have demonstrated that angiogenesis in acellular nerve allografts (ANAs) can promote neuroregeneration. The present study aimed to investigate the microvascular regeneration pattern of ANAs in Sprague-Dawley (SD) rats. Methods: Sixty male SD rats were randomly divided into an autologous group and a rat acellular nerve allograft group (rANA), and 10-mm sciatic nerve defects were induced in these rats. On the 7th, 14th and 21st days after surgery, systemic perfusion with Evans Blue (EB) or lead oxide was performed on the rats through carotid intubation. Samples were then collected for gross observation, and the microvessels in the nerves were reconstructed through microscopic CT scans using MIMICS software. The vascular volume fraction (VF, %) and microvessel growth rate (V, mm/d) in both groups were then measured, and 1 month after surgery, NF-200 staining was performed to observe and compare the growth condition of the axons. Results: Early post-operative perfusion with gelatin/EB showed EB permeation around the acellular nerve. Perfusion with gelatin/lead oxide showed that the blood vessels had grown into the allograft from both ends 7 days after the operation. Fourteen days after the operation, the microvessel growth rate of the autologous group was faster than that of the rANA group (0.39 ± 0.17 mm/d vs. 0.26 ± 0.14 mm/d, p < 0.05), and the vascular VF was also higher than that of the rANA group (8.92% ± 1.54% vs. 6.31% ± 1.21%, p < 0.05). Twenty-one days after the operation, the blood vessels at both ends of the allograft had connected to form a microvessel network. The growth rate was not significantly different between the two groups; however, the vascular VF of the autologous group was higher than that of the rANA group (12.18% ± 2.27% vs. 9.92% ± 0.84%, p < 0.05). One month after the operation, the NF-200 fluorescence (IOD) in the autologous group significantly increased compared with that of the rANA group (540,278 ± 17,424 vs. 473,310 ± 14,636, respectively, p < 0.05), suggesting that the results of the repair after nerve injury were significantly better in the autologous group than in the rANA group. Conclusion: Both the autologous nerve and ANAs rely on the permeation of tissue fluids to supply nutrients during the early stage, and microvessel growth mainly starts at both ends of the graft and enters the graft along the long axis. Compared to ANAs, the growth speed of revascularization in autologous nerve grafts was faster, leading to a better outcome in the autologous nerve group.

Etifoxine promotes glia-derived neurite outgrowth in vitro and in vivo.

BACKGROUND  Peripheral nerve injuries usually require a graft to facilitate axonal regeneration into the distal nerve stump. Acellular nerve grafts are good candidates for nerve repair, but clinical outcomes from grafts are not always satisfactory. Etifoxine is a ligand of the 18-kDa translocator protein (TSPO) and has been demonstrated to serve multiple functions in nervous system. METHODS  This study aimed to determine the optimal concentration of etifoxine for neurite outgrowth using PC12 cells and verify whether etifoxine could enhance in vivo peripheral nerve regeneration. PC12 cells were exposed to various concentrations of etifoxine (5, 10, 20, and 40 µM). Neuronal-like outgrowth and glia-derived neurotrophic factor (GDNF) mRNA expression were measured, and a rat sciatic nerve transection model was employed. Histological examination was used to evaluate the efficacy of nerve regeneration, and real-time polymerase chain reaction (PCR) evaluated changes in mRNA levels after etifoxine treatment. RESULTS  Our data show that etifoxine increased neuronal-like outgrowth in PC12 cells in a dose-dependent manner; however, GDNF expression peaked at 20 µM etifoxine (1.97-fold increase compared with control, p = 0.0046). In vivo studies demonstrated that etifoxine improved sciatic nerve regeneration, modulated immune responses, and boosted neurotrophin expression. CONCLUSIONS  Because of etifoxines adverse effects, we suggest an optimal etifoxine concentration of 20 µM. Its beneficial role may lie in increased neurotrophin expression, and etifoxine may be a promising therapeutic for patients with peripheral nerve injuries.