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. 2014 Oct 16;35(2):273–282. doi: 10.1007/s10571-014-0122-1

Ginkgo Biloba Extract (EGb 761) Promotes Peripheral Nerve Regeneration and Neovascularization After Acellular Nerve Allografts in a Rat Model

Zhaowei Zhu 1, Xiang Zhou 1, Bo He 1, Ting Dai 2, Canbin Zheng 1, Chuang Yang 3, Shuang Zhu 1, Jiakai Zhu 1, Qingtang Zhu 1,✉, Xiaolin Liu 1,✉
PMCID: PMC11486258  PMID: 25319407

Abstract

This study aimed to investigate whether or not ginkgo biloba extract (EGb 761) enhances peripheral nerve regeneration and vascularization after repair using acellular nerve allografts (ANA). Seventy-two Sprague–Dawley rats were randomly divided into three experimental groups: a unilateral 15-mm sciatic nerve defect was created and repaired with an autologous graft (autograft group); the same defect was repaired with an 18 mm ANA with an i.p. injection of normal saline for 10 days (saline group); and in the final group, the same defect was repaired with an 18 mm ANA with an i.p. injection of EGb 761 for 10 days (EGb 761 group). Axon outgrowth and vascularization were evaluated by immunocytochemistry 14 days post-implantation. The expression of genes associated with angiogenesis was analyzed by real-time polymerase chain reaction (PCR) seven days post-implantation. Compared with the saline group, rats in the EGb 761 group significantly increased the number of myelinated fibers and the average diameter of the nerves within the graft. There is no significant difference between the EGb 761 group and the autograft group. The expression of CD34 and NF200 was significantly higher in the EGb 761 group than in the saline group. Additionally, EGb 761 treatment increased the expression of several angiogenesis-related genes, including Vegf, SOX18, Prom 1, and IL-6. In conclusion, ANA repair with EGb 761 treatment demonstrates effects on peripheral nerve regeneration and vascularization that are equal to those of autologous graft repair, and that are superior to ANA repair alone.

Electronic supplementary material

The online version of this article (doi:10.1007/s10571-014-0122-1) contains supplementary material, which is available to authorized users.

Keywords: EGb 761, Acellular nerve allografts, Nerve regeneration, Angiogenesis

Introduction

Traumatic injury to peripheral nerves causes considerable loss of sensory and motor function, resulting in decreased quality of life. Despite advances in microsurgical techniques, the results of surgical repair are still less than ideal (Yu et al. 2009). Autologous nerve grafting is commonly used for nerve reconstruction; however, this technique has some major shortcomings which include a shortage of donor nerve material for repair and the requirement for the sacrifice of donor nerves especially in rare instances where the individual needing a nerve graft cannot provide donor material from a healthy nerve.

Acellular nerve allografts (ANAs) were derived from native peripheral nerves, so it could retain the basic structural and extracellular matrix (ECM) components of the original nerve, and once the cell component was removed, it stimulate only a low host immune response. Our group has demonstrated the effectiveness of ANAs for the repair of nerve defects in animal models (Wang et al. 2008; Zhou et al. 2013a, b). Furthermore, ANAs showed promising results in trauma patients with nerve defects in an ongoing multicenter clinical trial and with our technology, we can now produce many ANAs from one donor (He et al. 2013). However, there remain some problems. The efficiency of nerve regeneration and functional recovery in the ANAs is low (Yu et al. 2009; Walsh et al. 2009). Various substances have been studied with which to enhance peripheral nerve regeneration, but few of them have focused on neovascularization. Therefore, efforts need to be made to improve angiogenesis in implanted scaffolds, as well as nerve regeneration.

Angiogenesis, the formation of new blood vessels from the existing vascular system (Gerwins et al. 2000; Nguyen et al. 2001) is crucial for the regeneration of neural tissues. Appropriate angiogenesis in the implanted scaffold prevents exclusion of the graft via the formation of a fibrous capsule around the biomaterial, and helps to hold the implant in place (Friedman et al. 2002). In addition, angiogenesis provides nutrition and oxygen to growing tissues, as well as transplanted stem cells. Stem cell transplantation is proving to be one of the most promising biological advances for therapeutic strategies; however, the poor survival of transplanted stem cells has impeded its application in the field of peripheral nerve injury and regeneration. Therefore, appropriately vascularized scaffolds are more likely to support transplanted cells. Our group previously proved that acellular nerve grafts are effective in repairing small and large defects in animal models, so efforts need to be made to improve angiogenesis in implanted scaffolds.

In recent years, herbal remedies have been increasingly considered to be effective and safe alternatives to synthetic drugs in the industrialized world. However, the acceptance of herbal remedies is still in its infancy because of inadequate standardization and the lack of quality specifications. One of the few well-established plant products is the standardized special extract EGb 761 from the leaves of G. biloba. This extract has been demonstrated to be effective in clinical studies for the treatment of cerebral and peripheral vascular insufficiency, dementia (Ernst et al. 2009), diseases with a vascular background, intermittent claudication (Pittler and Ernst 2000), Raynaud’s disease (Muir et al. 2002), and tinnitus (Morgenstern and Biermann 2002). EGb 761 and its constituents, particularly terpenoids and flavonoids, have also been reported to possess vasorelaxant properties. EGb 761 has also been reported to enhance neurogenesis in the central nerve system (Yoo et al. 2011; Tchantchou et al. 2007; Tchantchou et al. 2009). In addition, EGb 761 possesses the ability to enhance nerve regeneration in the peripheral nervous system. Using a sciatic nerve injury model, Lin et al. observed that ginkgo biloba extract has the effect of promoting regeneration of injured peripheral in a dose-dependent manner (Lin et al. 2007). In a PLGA conduits repair sciatic nerve model, the addition of EGb 761 to a Schwann cell-seeded conduit increased the total number of myelinated axons in the regenerated nerve and improved functional recovery (Hsu et al. 2004).

However, the role of EGb 761 in peripheral nerve regeneration and vascularization after ANA implantation is unknown. Therefore, in this study, we tried to investigate if EGb 761 can enhance sciatic nerve regeneration after ANA repair.

Materials and Methods

Surgical Procedures

This study was approved by the Experimental Animal Administration Committee of Sun Yat Sen University. Efforts were taken to minimize animal suffering during the experiment.

Male SD rats (180–250 g) were anesthetized with an intraperitoneal injection of 10 % Chloral hydrate (10 ml/kg). Procedures were followed from other published article. Under aseptic conditions, the skin of the left leg was cut parallel to the femur, and the sciatic nerve was exposed by splitting the superficial gluteus muscle (Zhou et al. 2013a, b). With the aid of a surgical microscope, a 15-mm segment of the left sciatic nerve was severed and removed near the obturator tendon in the mid-thigh (Wilson et al. 2010; Rustemeyer et al. 2010). Animals were randomly divided into three groups: (1) the autograft group (n = 24); (2) the saline group (n = 24); and (3) the EGb 761 group (n = 24). In autograft group, a 15-mm segment of the sciatic nerve was removed, inverted, and reimplanted into the gap; in saline group, a 15-mm segment of the sciatic nerve was replaced by an 18 mm ANA, with daily i.p. injections of 0.5 ml/100 g vehicle (0.9 % NaCl solution) for 10 days; and in EGb 761 group, a 15-mm segment of the sciatic nerve was replaced by an 18 mm ANA, with daily i.p. injections of EGb 761 (50 mg/kg; Si-Te New Tech Co, Guilin, China) for 10 days. Previous studies have shown that IP saline injection has no any effect on nerve regeneration and could be used as a good control (Wilson et al. 2010; Pan et al. 2009). The muscle and skin were sutured with 6-0 and 4-0 polyamide sutures. The treatment of drug was started 24 h after the surgery.

ANA Preparation

For acellular nerve graft preparation, 12 Wister rats were sacrificed by intraperitoneal injection with sodium pentobarbital (0.5 ml, 60 mg/ml). The bilateral sciatic nerves were excised as much as more, then external debris was cleaned, and treated with chemical reagent to yield ANAs. The method for isolating acellular grafts was based on a procedure developed by Sondell et al. (Sondell et al. 1998).

Immunohistochemistry

Two weeks after surgery, in each group six rats were anesthetized with 10 % Chloral hydrate (10 ml/kg) and perfused thoroughly with saline, followed by 4 % paraformaldehyde. The sciatic nerves were taken out, post-fixed in 4 % paraformaldehyde for 3 h, and stored in 30 % sucrose (in 0.1 M PB) overnight at 4 °C. A cryostat was employed to cut 10-μm-thick sections of the middle part of the nerve, and then they were mounted on slides and subjected to immunohistochemistry with antibodies against CD34 and neurofilament 200 (NF-200) (Sigma-Aldrich, Tokyo, Japan). The secondary antibodies used were goat anti-mouse conjugated to cyanine-3 (Affiniti, UK; 1:200) and goat anti-rabbit FITC (TCS Biologicals, UK; 1:200). Antibodies were diluted in PBS solution containing 3 % rat serum, 3 % goat serum, and 0.02 % sodium azide (BDH Chemicals, UK) to reduce background staining.

For image study, photos were taken under the same condition (the same exposure time: 35 ms, the same filter). Computerized image analysis (Seescan Analytical Services, Cambridge, UK) was used to assess regenerated axon and blood vessels within the graft. For the determination of neurofilament and CD34 expression, six nerves in each group were cut longitudinally into 10-μm-thick sections and stained with each antibody. The middle parts of the grafts were chosen to be examined. The integrated optical density (IOD) of positive staining was assessed using Image-Pro Plus. For each animal and for each stain, calculations were made on three randomly chosen sections, and the results were averaged.

HE Staining

Four weeks after surgery, six rats per group were killed for histological examination. The middle part of gastrocnemius muscles was harvested from all three groups. The muscle samples were post-fixed with paraformaldehyde, embedded in paraffin, and sectioned transversely. Then, the sections were stained with hematoxylin and eosin. For each sample, photos were taken from three random fields, and Image-Pro Plus (Version 6.0) software was used to measure the area of the muscle. Six samples from each group were used for statistical analysis. Relative muscle area was calculated by normalizing the collateral normal muscle.

Toluidine Blue Staining

Four weeks after surgery, middle part of the repaired nerve segments was harvested from the animals sacrificed for Toluidine blue staining. Toluidine blue staining was performed as previously described (Zhou et al. 2013a, b). Briefly, nerve graft segments were harvested and quickly immersed in 2.5 % Na-cacodylate-buffered glutaraldehyde solution for 2 h, fixed for 2 h in 2 % Na-cacodylate-buffered osmium tetroxide, serially dehydrated in increasing concentrations of ethanol, infiltrated with and embedded in Epon 812 (Ted Pella, Redding, CA, USA), sectioned (1 μm thick), and stained with toluidine blue to evaluate the efficacy of nerve regeneration (Zhou et al. 2013a, b). Transection was made in the middle of the nerve graft. Analysis of the average number of myelinated axons and fiber diameter was performed using an Olympus BX60 microscope and Image-Pro Plus (Version 6.0) for quantitative morphological study. For each of the sample, photos were taken from three random fields, and analyzed to measure the number of axons as well as their diameters. Six samples from each group were used for statistical analysis.

Electron Microscopy

Four weeks after surgery, six rats per group were sacrificed for electron microscopy. We use transmission electron microscopy to estimate myelin sheath regeneration. Ultrathin sections were stained with lead citrate and uranyl acetate, and then examined under a Philips CM120 transmission electron microscope equipped with an image acquisition system with 8,000× magnification to measure the thickness of the myelin sheaths according to the previous report (Zhou et al. 2013a, b). Photographs from 6 random fields of each section were analyzed using Image-Pro Plus.

Real-Time qPCR Analysis

One week after surgery, 6 rats per group were sacrificed, and quantitative real-time PCR was used to determine the mRNA levels of SRY-box containing gene 18 (Sox18), Prominin 1 (Prom1), vascular endothelial growth factor (Vegf), activin A receptor type II-like 1 (Acvrl1), and interleukin 6 (IL-6) in the distal nerve segment. β-actin was used as a control. Primers for rat Sox18, Prom1, Vegf, Acvrl1, β-actin, and IL-6 were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA), and were obtained from Shenggong Bio Technologies (Shanghai, China) (Supplementary Table 1). Twelve rats were sacrificed, six from the saline group and six from the EGb 761 group, and their sciatic nerves were stored in RNA-Later (Ambion, Austin, TX) at −20 °C. Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA), purified on RNeasy minicolumns (Qiagen, Valencia, CA), and treated with RNase-free DNAse I (Qiagen). The RNA purity (OD260/280 absorption ratio) was approximately 1.9–2.0. cDNA was synthesized using a SuperScript II first-strand RT-PCR kit (Invitrogen). Gene expression was measured by qPCR (MX4000, Stratagene, La Jolla, CA) with 50 ng of rat cDNA and 2 x Taqman universal PCR master mix (Applied Biosystems) with a one-step program (95 °C for 10 min, 95 °C for 30 s, and 60 °C for 1 min for 50 cycles). Duplicate samples without cDNA (no template control) for each gene showed no contaminating DNA. Gene expression levels were normalized to β-actin and were quantified using the comparative critical threshold (Ct) method. The PCR primers are in supplementary data.

Statistical Analysis

All numerical data are given as mean ± standard error (SE). Statistical analysis was performed using SPSS 11.5 software for Windows (student version). Group comparisons were performed using ANOVA, and pair-wise comparisons were made by post-hoc tests (Bonferroni correction). Statistically significant values were defined as P < 0.05.

Results

EGb Treatment Increases Density of Regenerated Axons

Two weeks after surgery, longitudinal sections in the middle of the sciatic nerves were examined quantitatively to assess the density of regenerated axons. Six rats from each group were sacrificed, and the expression of neurofilament protein, the increase of which reflects early regenerative potential, was measured by immunohistochemistry (Omura et al. 2004). Analysis of IOD (Fig. 1) demonstrated that the expression of neurofilament protein was significantly higher in the autograft group and the EGb 761 group, as compared to the saline group (530,015 ± 24,316 vs. 470,907 ± 23,854) (P < 0.01) .

Fig. 1.

Fig. 1

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Immunohistochemistry analysis of longitudinal sections in the middle of the bridge two weeks after surgery Repair using a autologous sciatic nerve, b ANA combined with EGb 761 treatment, and c ANA with normal saline. d Normal sciatic nerve, e fiber counts by examining neurofilament expression demonstrate significantly greater numbers of nerve fibers in the autologous group and EGb 761 group than in the saline group. Error bars indicate mean ± SE. *P < 0.05

EGb Treatment Increases the Muscle Mass

Atrophy in the rat gastrocnemius muscle as a result of sciatic nerve damage was assessed by H&E staining 4 weeks post-surgery. Analysis of the muscle atrophic process reveals the gradual recovery of sciatic nerve function (Wang et al. 2005). Muscles from the saline groups presented smaller muscle cell sectional area as compared with the muscles from the autograft and EGb 761 groups (Fig. 2). The contralateral gastrocnemius muscles with an intact nerve and no muscle atrophy were used as controls to calculate relative muscle area. EGb 761 treatment significantly increased the average percentage of muscle fiber area (71.3 ± 1.3 % vs. 66.4 ± 3.1 %, P < 0.01), thus showing that better nerve regeneration following EGb 761 treatment.

Fig. 2.

Fig. 2

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H&E staining of gastrocnemius muscle a autograft group. b EGb 761 group. c Saline group. d Normal group. e Relative muscle area. Relative muscle area was calculated by normalizing the collateral normal muscle. Compared with the saline group, EGb 761 greatly increased the relative muscle area. Error bars indicate mean ± SE. *P < 0.05

EGb Treatment Increases Axon Number and Diameter

To examine the diameter and number of axons, cross-sections in the middle of the grafts were stained with toluidine blue four weeks post-surgery. Sections from the EGb 761 group and the autograft group demonstrate populations of axons that successfully regenerated in the grafts (Fig. 3). In the autograft group, myelinated fibers were of similar size and shape and were symmetrically arranged; in the EGb 761 and saline groups, myelinated fibers were randomly spaced and less symmetrically shaped.

Fig. 3.

Fig. 3

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Histological sections of regenerated nerves. Semi-thin cross-sections of the distal portion of each nerve graft were stained with toluidine blue four weeks post-surgery. a Intact rat sciatic nerve. b Autograft group. c EGb 761 group. d Saline group. e Average diameter of the nerve. f Number of myelinated axons. Thin (1 μm) sections of sciatic nerve specimens were stained with 1 % toluidine blue for qualitative examination of the midline of the bridge by light microscopy. Groups with EGb 761 demonstrated more organized neural architecture, closely approximating the autograft, in comparison to the saline group. Error bars indicate mean ± SE. Significance of differences was determined by t test. Results were obtained from six rats in each group. *P < 0.05

No significant difference was observed in the number of axons between the EGb 761 group and autograft group, suggesting that regeneration occurred equally in both groups (48,321 ± 1,307 vs. 53,876 ± 2,382; P > 0.05). The number of myelinated (toluidine blue positive) axons was significantly lower in the saline group as compared with the EGb 761 group (40,143 ± 1,562 vs. 48,321 ± 1,307, P < 0.001) (Fig. 3f). Further comparison of the average diameter of the myelinated fibers did not reveal any significant difference between the EGb 761 group and the autograft group (2.65 ± 0.32 vs. 2.75 ± 0.31; P > 0.05); however, the average diameter of the myelinated fibers was significantly lower in the saline group than in the EGb 761 group (1.93 ± 0.15 vs. 2.65 ± 0.32, P < 0.05) (Fig. 3e). One month after implantation, transmission electron micrographs were acquired to assess the ultrastructure of regenerated myelinated fibers in the mid-portion of the graft. Results show that the myelin sheath thickness was greater in the EGb 761 group than in the saline group (0.94 ± 0.10 vs. 0.67 ± 0.12, P < 0.05), but myelin sheath thicknesses were similar in the EGb 761 and autograft groups (Fig. 4d).

Fig. 4.

Fig. 4

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Transmission electron micrographs a Autograft group. b EGb 761 group. c Saline group. d Myelin sheath thickness of each group. Error bars indicate mean ± SE. *P < 0.05

EGb Treatment Increases Axon Angiogenesis

Immunofluorescent staining for CD34, a marker of axon angiogenesis, was performed on regenerating nerves two weeks after surgery. We found that CD34 expression was significantly increased in the autograft and EGb 761 groups as compared to the ANA group (85.6 ± 8.5, 84.4 ± 9.3 vs. 77.0 ± 6.4; P < 0.05). No significant difference was observed in the expression of CD34 between the autograft group and the EGb 761 group (77.0 ± 6.4 vs. 64.1 ± 4.1; P > 0.05) (Fig. 5d). Taken together, these results suggest that EGb 761 treatment significantly promotes axon angiogenesis in ANA-repaired animals.

Fig. 5.

Fig. 5

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Axon angiogenesis stained by CD34. Immunohistochemistry analysis of longitudinal sections in the middle of the bridge two weeks after surgery repair using a Autologous sciatic nerve, b ANA repair combined with EGb 761 treatment, and c ANA repair with normal saline. d Normal sciatic nerve. e CD34 expression. CD34 expression was greater in the autologous group than the EGb 761 group, and greater in the EGb 761 group than the saline group. Error bars indicate mean ± SE. *P < 0.05

Further evaluation of the expression of angiogenesis-related genes in the regenerating sciatic nerve revealed that the mRNA levels of Sox18, Prom1, Vegf, and IL-6 significantly increased after the EGb 761 treatment as compared to the saline-treated group, while no change of the expression of those genes was observed between the autograft and EGb 761 groups (Fig. 6). The expression of Sox18 was remarkably higher in the autograft group than in the EGb 761 group.

Fig. 6.

Fig. 6

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Effects of EGb 761 on angiogenesis-associated gene mRNA expression mRNA levels in nerves were normalized to β-actin mRNA levels. Data are expressed as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the EGb 761 (ANA+E) and ANA groups. Error bars indicate mean ± SE

Discussion

Ginkgo biloba extract is an extract from the leaves of Ginkgo biloba, a unique tree originating from Asia, mainly Southeast China. EGb 761, a standardized preparation of the extract (Dr. Willmar Schwabe Pharmaceuticals, Karlsruhe, Germany), has proved to be useful in treating cerebral and peripheral vascular insufficiency. EGb 761 and its constituents, particularly terpenoids and flavonoids, have also been reported to possess vasorelaxant properties (Duarte et al. 2001; Ibarra et al. 2003). Clinical studies have shown that EGb 761 exhibits therapeutic activity in a variety of CNS and cardiovascular diseases (Gertz and Kiefer 2004). EGb 761 has also been reported to promote the regeneration of injured peripheral nerves dose-dependently (Lin et al. 2007).

Considering the effect of EGb on the vascular system and on nerve regeneration, we investigated the efficacy of EGb 761 on peripheral nerve regeneration and vascularization in a rat sciatic nerve injury model after ANA repair. In our study, the degree of axonal regeneration was significantly higher in the EGb 761 group than in the saline group. Previous reports show that muscles may undergo irreversible atrophy after nerve damage and lose function before axons can regenerate into their distal targets. Therefore, the speed of regeneration is important to the recovery of muscle function. Our results showed that in the early stage of nerve regeneration, EGb 761 treatment accelerated nerve regeneration after ANA repair. The ability for regenerating peripheral axons to reach muscular end plates before atrophy would be very beneficial. In this study, the atrophy of the gastrocnemius muscles was comparable to the autograph group, when EGb 761 treatment was provided after acellular graft implantation.

Histological assessment showed that nerve fibers grew better in the EGb 761 group, and the structure of the regenerated nerves was similar to that of the autograft group (Fig. 3). In contrast, the myelinated nerve fibers in the saline group were less dense, of uneven size, and less myelinated. H&E staining revealed that three months after implantation, gastrocnemius muscle atrophy was the most obvious in the saline group, suggesting little recovery of nerve function. However, the recovery level in rats treated with EGb 761 approached that of the autograft group.

Interestingly, in addition to axon growth, angiogenesis was detected after ANA repair, which was further enhanced by EGb 761 treatment. The expression of several angiogenesis-related genes, including Vegf, SOX18, Prom 1, and IL-6, was examined by Quantitative RT-PCR. Vegf is an angiogenic factor that is critical for vascularization/angiogenesis, and its expression is reported to be elevated in injured sciatic nerves (Pola et al. 2004). Studies also show that increased Vegf expression can augment angiogenesis and increase vessel density in vivo after injury (Jung et al. 2006; Sehara et al. 2007). Sox18 is an important regulator of vascular development, playing roles in endothelial cell specification, differentiation, and angiogenesis (Petrovic et al. 2010). Sox18 knockdown disrupted vasculogenesis and angiogenesis in zebrafish (Chung et al. 2011), and the Sox18 transcription factor is a key regulator of murine and human blood vessel formation (Young et al. 2006). Prominin 1 encodes a pentaspan transmembrane glycoprotein, which localizes to membrane protrusions and is often expressed in adult stem cells. Many studies have shown that Prom1 is closely related to angiogenesis. ACVRL1 encodes ALK1, an endothelial transforming growth factor β receptor that is involved in angiogenesis. Genetic and pharmacological targeting of activin receptor-like kinase 1 impairs angiogenesis (Cunha et al. 2010). Interleukins (ILs) play important roles in cancer as potential modulators of angiogenesis, leukocyte infiltration, and tumor growth. Saraswati et al. found that decreased levels of Vegf and IL-6 could suppress angiogenesis (Saraswati and Agrawal 2013). In our study, the expression levels of Sox18, Prom1, Vegf, and IL-6 increased significantly with EGb 761 treatment after ANA repair as compared with normal saline injection. These results suggest a possible explanation for increased angiogenesis by EGb 761 treatment.

Endothelial progenitor cells (EPCs) might also play a role in the vascular effect of EGb 761. EPCs are a class of precursor cells that can proliferate and differentiate into vascular endothelial cells, but do not express markers of mature vascular endothelial cells or form blood vessels (Asahara et al. 1997; Peichev et al. 2000). EPCs are involved not only in human embryonic angiogenesis, but also in vasculogenesis and the restoration of endothelial function following injury after birth. Approximately 25 % of the endothelial cells in the neovascularization differentiated from EPCs (Murayama et al. 2002; Suzuki et al. 2003). Incubation of isolated human MNCs with ginkgo biloba extract increased the number of EPCs and promoted EPC proliferation, migration, adhesiveness, and the capacity for in vitro vasculogenesis (Chen et al. 2004). However, this explanation is not tested in our study and needs additional investigation.

In conclusion, ANA repair with EGb 761 treatment demonstrates equal effects on peripheral nerve regeneration and vascularization when compared with the autologous graft repair, and superior effects compared to ANA repair alone. Considering the fact that this drug has already been widely used in clinical settings, incorporating EGb 761 in ANA repair might be a promising strategy in the treatment of peripheral nerve injury.

Electronic Supplementary Material

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Supplementary material 1 (DOCX 11 kb) (11.9KB, docx)

Acknowledgments

We are grateful to Weihong Yang for technical assistance. This study was supported by Grants from the National High Technology Research and Development Program of China (contract Grant Number: 2012AA020507), the National Nature Science Grant of China (30700847), the Combined Grant of Guangdong and Ministry of Education of China (2007B090400090), the Key Project of Nature Science Grant of Guangdong China (9251008901000017), and grants from the National Basic Research Program of China (973 Program, No. 2014CB542201).

Ethical Standards

All animal studies have been approved by The Institute Research Medical Ethics Committee of Sun Yat-Sen University.

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Zhaowei Zhu and Xiang Zhou have contributed equally to this article.

Contributor Information

Qingtang Zhu, Phone: +86-13502167619, Email: qtzhusci@qq.com.

Xiaolin Liu, Phone: +86-13600481606, Email: liuxiaolinsci@163.com.

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