Original Article WSRM 2013 Scientific Paper
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Etifoxine Promotes Glia-Derived Neurite Outgrowth In Vitro and In Vivo Ting Dai, PhD1, Xiang Zhou, PhD, MD2, Yanan Li, PhD3 Bo He, MD, PhD2 Zhaowei Zhu, MD, PhD2 Canbin Zheng, MD, PhD2 Shuang Zhu, MD, PhD2 Qingtang Zhu, MD, PhD2 Xiaolin Liu, MD, PhD2
Medical University, Guangzhou, China 2 Department of Microsurgery and Orthopaedic Trauma, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China 3 Department of Biochemistry & Molecular Biology, School of Basic Science, Guangzhou Medical University, Guangzhou, China,
Address for correspondence Xiaolin Liu, MD, PhD, 58 Zhongshan Er Road, Guangzhou, China (e-mail: [email protected]).
These authors contributed equally to this work. J Reconstr Microsurg 2014;30:381–388.
Abstract
Keywords
► etifoxine ► GDNF ► neurite outgrowth
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 ¼ 0.0046). In vivo studies demonstrated that etifoxine improved sciatic nerve regeneration, modulated immune responses, and boosted neurotrophin expression. Conclusions Because of etifoxine’s 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.
The most common method of repairing injured nerves is to directly suture two severed nerve ends together. However, this method is often impossible if the gap between the two nerve ends is too large to allow for tension-free nerve repair. Several research groups have tried to accelerate nerve regeneration through microsurgical techniques. However, despite recent ad-
vances in these techniques, complete recovery is rarely achieved.1 Acellular nerve allografts (ANAs) derived from native peripheral nerve retain the structure and extracellular matrix (ECM) components of the original nerve and stimulate little host immune response. Previously, our group demonstrated the usefulness of acellular nerve grafts to repair small and large
received October 24, 2013 accepted after revision April 8, 2014 published online June 23, 2014
Copyright © 2014 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0034-1381751. ISSN 0743-684X.
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1 Department of Biotechnology, School of Basic Science, Guangzhou
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nerve injuries in several animal models.2–4 On the basis of those findings, acellular nerve grafts have been attempted in humans, and a multicenter trial has been completed. Those results indicate that hANA is a safe and effective nerve repair technique for nerve injuries of 1 to 5 cm in size.5 However, because the efficiency and effective repair length of acellular nerve grafts are still low,3,5–7 a promising therapeutic approach could involve combining nerve grafts with techniques to enhance nerve cell regeneration within the graft. Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H3, 1-benzoxazine hydrochloride; Stresam, [Gentilly, Val-deMarne, France], Biocodex) is currently under investigation as a promising therapeutic to promote neuroprotection, accelerate axonal regeneration, and modulate inflammation.8,9 Traditionally, etifoxine is used as an anxiolytic and anticonvulsant drug. Recent research showed that it targets GABA A receptors and the 18-kDa translocator protein (TSPO).10 In addition to etifoxine, Mills et al found that Ro5–4864, another TSPO ligand, significantly enhanced regeneration and functional recovery in the rat peripheral nervous system.11 Zhou et al speculated that TSPO activation will lead to an increase of steroidogenesis and that increased steroids may affect glia-derived neurotrophic factor (GDNF) gene expression through cAMP-response element binding protein (CREB) phosphorylation. Moreover, etifoxine stimulated GDNF-induced neurite outgrowth in PC12 cells,12 but details surrounding optimal therapeutic etifoxine concentrations are still lacking. In this study, we used a well-defined PC12 cell model to test the effects of etifoxine on GDNF-induced neurite outgrowth. Then, we verified its effects using a 10-mm rat sciatic transection model. We found that etifoxine leads to increased neurite outgrowth at an optimal concentration of 20 µM. In addition, in vivo experiments showed that the etifoxinetreated group experienced enhanced nerve fiber growth, and the structure of regenerated nerves was similar to the regenerated nerve structure in the autograft group. These data demonstrate a potential therapeutic role for etifoxine in human patients.
Methods Cell Culture and Neurite Outgrowth Measurements Rat PC12 cells were cultured on collagen-coated plates (5 µg/cm2) in Dulbecco Modified Eagle Medium supplemented with 5% horse serum, 10% FBS, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Etifoxine dissolved in dimethyl sulfoxide (5, 10, 20, and 40 µM final concentration), and then it was added to cultures. Neuronal-like outgrowth was determined after 10 days of treatment using an Olympus IX81 microscope and U-CMAD 3 camera (Olympus, Tokyo, Japan). Cells were counted in six fields of view of the cell culture dish. At least 400 cells were counted per sample, and experiments were repeated at least three times. Cell axon length was analyzed using image analysis software, version 3.2 (SIS, Münster, Germany). PC12 cells with axons longer than the average cell diameter were used for data analysis. The two-tailed Mann–Whitney U-test (GraphPad Prism 4; GraphPad Software, San Diego, CA) was used to Journal of Reconstructive Microsurgery
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assess statistical significance of GDNF release and its bioactivity on PC12 cell axonogenesis.
Real-Time Polymerase Chain Reaction Real-time polymerase chain reaction (RT-PCR) was performed as previously described.12 Total RNA was extracted from 1 106 cells. First strand cDNA was synthesized from total RNA (1 mg) using avian myeloblastosis virus reverse transcriptase (RT) XL primed by 50 pmol of random 9-mers (TaKaRa Biomedicals, Tokyo, Japan). The RT reaction products (10 µL) were utilized as templates in PCR with 0.2 mM of each of the following primers: GDNF, forward 5′-GGTCTACGGAGAGACCGATCCGAGGTGC- 3′ and reverse 5′-TCTCTGGAGCCAGGGTCAGATACATC-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-TGAAGGTCGGTGTCAACGGATTTGGC-3′ and reverse 5′-CATGTAGGCCATGAGGTCCACCAC- 3′. PCR products were separated by electrophoresis with precast 2% agarose gels (Daiichi Pure Chemicals, Tokyo, Japan) and visualized by SYBR Green 2 (FMC BioProducts, Rockland, ME) staining and a UV transilluminator. The PCR product signal intensities were determined with ImageJ, and the amount of GDNF PCR product was determined by normalizing to the GAPDH signal intensity.
Surgical Procedures A total of 50 male Sprague-Dawley rats (range, 200–230 g) were used in this study. In addition, six male Wistar rats were used for nerve grafts. All animal procedures were approved by the Experimental Animal Administration Committee of Sun Yat-sen University, China. Efforts were made to minimize pain and suffering during the postsurgery period. Animals were randomly divided into the following three groups: (1) autograft group (n ¼ 18), where a 10-mm sciatic nerve segment was removed, inverted, and reimplanted into the surgical gap; (2) simple acellular nerve graft group (n ¼ 18), where a 10-mm sciatic nerve segment was replaced by a 12-mm acellular nerve graft, and daily intraperitoneal (i.p.) injections of 0.5 mL/100 g vehicle (1% Tween-80 in 0.9% NaCl solution) were administered; and (3) acellular nerve graft plus etifoxine group (n ¼ 18), where daily i.p. etifoxine injections (Batch 285; Biocodex, Gentilly, France) were administered at a dosage of 50 mg/kg for 7 days. The 50 mg/kg dosage was selected because it had been previously shown to exhibit anxiolytic-like effects in rats.8 Male Sprague-Dawley rats (range, 200–230 g) were deeply anesthetized by intraperitoneal pentobarbital injection (75 mg/ kg), and surgical procedures were performed under deep anesthesia (40 mg/100 g of chloral hydrate) and aseptic conditions. The animals were monitored up to 4 weeks postsurgery. Rats were allowed free access to food and water ad libitum. To prepare nerve grafts, Wistar rats were euthanized by intraperitoneal sodium pentobarbital injection (0.5 mL, 60 mg/mL). The sciatic nerves were excised, cleansed of external debris, and treated with chemical detergents to produced acellular allografts. This method for isolating acellular grafts was derived from the method developed by Sondell et al. 13
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Four weeks post-surgery, rats were euthanized, and tissues were prepared for histological examination. The repaired nerve segments were harvested from all the three groups. Toluidine blue staining was performed as previously described.3 Briefly, nerve segments were harvested and quickly immersed in a 2.5% Na-cacodylate-buffered glutaraldehyde solution for 2 hours and fixed for 2 hours in 2% Na-cacodylatebuffered osmium tetroxide. Then, the nerve segments were serially dehydrated in increasing concentrations of ethanol, infiltrated and embedded in Epon 812 (Ted Pella, CA). A 1 µm cross-section of the nerve segment was obtained and then stained with toluidine blue to evaluate the efficacy of nerve regeneration. Analysis of the average number of myelinated axons and the fiber diameter was performed using an Olympus BX60 (Olympus, Melville, NY, USA) microscope and the Image-Pro-Plus (Version 6.0) quantitative morphology software. For each sample, images were acquired from three random fields, and the number of axons and the nerve fiber diameters were measured. Six samples from each group were tested for statistical significance. Transmission electron microscopy was employed to evaluate myelin sheath regeneration. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with a Philips CM120 (Philips, Eindhoven, Netherlands)
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transmission electron microscope equipped with an image acquisition system (8,000 magnification) to measure myelin sheath thickness. Images from 10 random fields of each ultrathin nerve section were analyzed with the ImagePro Plus software.
Quantitative Real-Time Polymerase Chain Reaction In nerve segments, quantitative real-time PCR was used to detect and measure mRNA levels of nerve growth factor (NGF), GDNF, vascular endothelial growth factor (VEGF), tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). Primers for rat NGF, GDNF, VEGF, TNF-α, IL-1, IL-6, and GAPDH were designed using the Primer Express 2.0 software (Applied Biosystems, Foster City, CA) and purchased from Huirui Bio Technologies (Shanghai, China). One week postsurgery, six rats per group were humanely sacrificed, and their sciatic nerves were stored in RNA-Later (Ambion, Austin, TX) at 20°C until use. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA), purified on RNeasy minicolumns (Qiagen, Valencia, CA), and treated with RNase free DNAse I (Qiagen , Valencia, CA). The RNA purity (OD260/280 absorption ratio) was measured at 1.9 to 2.0. cDNA was synthesized using a SuperScript II first-strand RT-PCR kit (Invitrogen , Carlsbad, CA). Gene expression was measured by qPCR (MX4000; Stratagene, La Jolla, CA) with 50 ng of rat cDNA and TaqMan Fast
Fig. 1 (A) Microscopic view of PC12 cells in saline, (B) 5 µM, (C) 10 µM, (D) 20 µM, and (E) 20 µM Etifoxine, 10 days of cultivation, figures demonstrating significant increase of axonal sprouting activity. (F) Statistics analysis shows that increase of average axon length was a dosagedependent increase model. A two-tailed Mann–Whitney U-test was used for statistical assessment. p < 0.05, p < 0.01.
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Histological Examination
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Universal PCR Master Mix (2) (Applied Biosystems) with a one-step program (50 cycles of 95°C for 10 minutes, 95°C for 30 seconds, and 60°C for 1 minute). For each gene, duplicate samples without cDNA (no template control) demonstrated the lack of contaminating DNA. Gene expression levels were normalized to GAPDH and quantified by using the comparative critical threshold (Ct) method.
Statistical analysis of PC12 cell neuronal-like outgrowth revealed highly significant etifoxine-induced axonogenesis, even when comparing 5 µM etifoxine (p ¼ 0.43; 28.25 3.43 µm) with the saline control (20.22 3.29 µm) (►Fig. 1F). The average axon length increased in a concentration-dependent manner and peaked at 40 µM etifoxine, with an average length of 42.26 9.76 µm.
Walking-Track Analysis
Etifoxine Induces GDNF mRNA Expression
Functional nerve regeneration was assessed by calculating the sciatic functional index (SFI) (de Medinaceli et al 1982). In total, 18 rats had been used to assess walking-track behavior. Briefly, after their hind feet were dipped in dark ink, rats walked down a wooden corridor. SFI was calculated using the following formula:
To address the mechanism of etifoxine-induced neuronal-like outgrowth in PC12 cells, we measured GDNF mRNA expression by RT-PCR using GDNF-specific primers as described earlier. After etifoxine treatment, GDNF mRNA expression levels increased by 1.50-fold (p ¼ 0.035) after 5 µM etifoxine treatment compared with the saline treatment (►Fig. 2). These data support the finding that etifoxine induces neuronal-like outgrowth in PC12 cells. Next, we determined the optimal etifoxine concentration that produced the largest induction of GDNF expression. PC12 cells were treated with increasing concentrations of etifoxine, and GDNF expression increased in a concentration-dependent manner from 5 to 20 µM. However, when 40 µM etifoxine was administered, GDNF expression decreased, but this change was not significant compared with 20 µM (1.97 0.23 and 1.91 0.26, p ¼ 0.95).
In this formula, the print length (PL) was defined as the distance from the heel to the third toe; the toe spread (TS) was defined as the distance from the first to fifth toe; and the intermediary toe spread (IT) was defined as the distance from the second to the fourth toe. All three measurements were made on the experimental (E) and normal (N) sides. The recovery of walking function is a gradual process after the surgery. To see the changes along this process, the walking functional test was performed every 4 weeks, beginning on the 4th week and ending on the 12th week.
Histological Examination After examining toluidine blue-stained nerve segments at 8 weeks postsurgery, etifoxine treatment enhanced axon
Statistical Analysis All the results were subjected to statistical analysis using SPSS 11.5 software (SPSS, San Francisco, CA) for Windows. For quantification in the article, two researchers read the results independently, final results come from the mean of those two researchers. Comparisons between two groups were performed by Student t-test. Statistically significant values were defined as p < 0.05. Statistically power was calculated using the software GPower 3.14 Power calculated for the cell study is 0.72. Power calculated for the animal study is 0.28.
Results Etifoxine induces neuronal-like outgrowth in PC12 cells in a dose-dependent manner. To examine whether etifoxine can promote GDNF-induced neurite outgrowth in PC12 cells, the length of outgrown fibers was measured. ►Fig. 1 clearly demonstrates the biological activity of etifoxine, and in agreement with previous reports showing etifoxine-induced neurite extension, a dramatic increase in neuronal-like outgrowth was observed 10 days after etifoxine application (►Fig. 1B). Next, to determine the optimal etifoxine concentration, cultured PC12 cells were treated with increasing concentrations of etifoxine (from 5 to 40 µM). In contrast to 5 µM etifoxine, higher etifoxine concentrations only increased PC12 cell neuronal-like processes after 10 days of treatment. Journal of Reconstructive Microsurgery
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Fig. 2 Expression of GDNF mRNA in drug-treated PC12 cells. The results of real time polymerase chain reaction analysis of GDNF mRNA in PC12 cells. (S, cell treated with saline. 5, cell treated with 5 µm Etifoxine. 10, cell treated with 10 µm Etifoxine. 20, cell treated with 20 µm Etifoxine. 40, cell treated with 40 µm Etifoxine.) A representative result is shown, and the data are presented as the mean and standard deviation of PC12 cell cultures in media (n ¼ 8). p < 0.05, p < 0.01, compared ANAþE with ANA. #p < 0.05, compared 20 µm with 40 µm, determined by two-tailed Student t-test. GDNF, glia-derived neurotrophic factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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Fig. 3 Histological sections of regenerated nerves. Semithin cross-sections of the distal portion of each nerve graft were stained with toluidine blue 4 weeks postsurgery. (A) Intact rat sciatic nerve. (B) Autograft group. (C) ANA group. (D) Etifoxine group. (E) Average diameter of the nerve. (F) Number of myelinated axons. Groups with etifoxine demonstrated more organized neural architecture, closely approximating the autograft, in comparison to the ANA alone group. Error bars indicate mean standard error of mean Significance of differences was determined by t-test (n ¼ 6). p < 0.05 compared with the etifoxine (ANA þ E) and autologous (auto) groups. ANA, acellular nerve allografts.
regeneration (►Fig. 3). Myelinated fibers were similar in size and shape and symmetrically arranged in the autograft group, but they were randomly spaced and less symmetrical in the etifoxine and ANA groups. The number and diameter of regenerated axons were larger in the etifoxine group than in the autograft group. The total number of axons was similar in the etifoxine and autograft groups, which suggests that axon regeneration was achieved in both the groups (49,537 2,123 vs. 57,523 2,023; p ¼ 0.12). The number of myelinated (toluidine blue positive) axons was significantly increased in the etifoxine group compared with the ANA group (39,105 2,387 vs. 57,523 2,023; p ¼ 0.031) (►Fig. 3E). Although similar in both the etifoxine and autograft groups (2.95 0.17 vs. 2.76 0.21), the average diameter of myelinated fibers was significantly larger in the etifoxine group compared with the ANA group (1.94 0.23 vs. 2.76 0.21; p ¼ 0.025) (►Fig. 3F). Along with histological evidence, transmission electron microscopy revealed regenerated myelinated fibers in the
mid portion of the implant 4 weeks after graft implantation in each group. The myelinated fibers were more compact and uniform in the etifoxine and autograft groups than the fibers of the ANA group. The average myelin sheath thickness of the etifoxine group was larger than the ANA group sheath thickness (1.07 0.17 vs. 0.65 0.13, p ¼ 0.028), but the average myelin sheath thickness was similar in the etifoxine and autograft groups (►Fig. 4D).
Gene Expression RT-PCR was utilized to investigate the potential changes in proinflammatory signals in response to peripheral nerve injury and etifoxine administration. The TNF-α transcript (1.11 0.14 vs. 0.64 0.13, p ¼ 0.034; ►Fig. 5) and IL-1 transcript (1.08 0.18 vs. 0.48 0.11, p ¼ 0.025) levels were reduced in the etifoxine group compared with the control group. Although etifoxine influences the immune response in rat sciatic nerve, it is unknown whether etifoxine influences neurotrophin expression in peripheral nerves. To determine Journal of Reconstructive Microsurgery
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Etifoxine Promotes Neurite Outgrowth
Etifoxine Promotes Neurite Outgrowth
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Fig. 4 Transmission electron micrographs. (A) Autograft group. (B) Acellular nerve allografts group. (C) Etifoxine group. (D) Myelin sheath thickness of each group. Error bars indicate mean standard error of mean Significance of difference was determined by t-test (n ¼ 6). p < 0.05.
whether etifoxine modulates neurotrophin expression after peripheral nerve injury, the expression levels of NGF, BDNF, and VEGF were quantified in regenerating sciatic nerves (►Fig. 5). At 7 days postinjury, etifoxine treatment had a marked neurotrophic effect (NGF, a 1.9-fold increase over control, p ¼ 0.019; GDNF, a 2.1-fold increase over control, p ¼ 0.0036; and VEGF, a 1.7-fold increase over control, p ¼ 0.072).
12 weeks postoperatively, functional recovery was almost the same in the etifoxine and autologous groups (►Fig. 6).
Discussion
Walking track analysis is a useful technique for evaluating motor functional recovery after sciatic nerve injury. The increase in sciatic nerve functional index after treatment indicates peripheral nerve regeneration. As shown in ►Fig. 6, SFI was significantly higher in the etifoxine group than the ANA group 8 weeks postoperatively. However, it is still lower than the autologous group. This result shows that etifoxine treatment enhances functional recovery in ANAs. By
In this study, we found that etifoxine-induced PC12 cell neuronal-like outgrowth, which increased up to 3.27-fold after 10 days and resulted in numerous partially arborescent axons. In addition, the optimal etifoxine concentration was 20 µM, which induced maximal stimulation of GDNF expression and neuronal-like outgrowth. In vivo experiments demonstrated that etifoxine-enhanced peripheral nerve regeneration across 10-mm nerve gaps repaired by acellular nerve grafts. Etifoxine is a TSPO ligand that associates with the mitochondrial permeability transition pore, which allows the electron transport chain to create the transmembrane electrochemical gradient (ΔΨm) driving ATP synthesis.15
Fig. 5 Effects of etifoxine on mRNA expression. mRNA levels in nerve were normalized to GAPDH mRNA levels. Data are expressed as the mean standard error of mean. Significance of difference was determined by t-test (n ¼ 6). p < 0.05, p < 0.01. ANA, acellular nerve allografts; GDNF, glia-derived neurotrophic factor; IL, interleukin; NGF, nerve growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
Fig. 6 Etifoxine treatment improved the functional recovery of transected sciatic nerve. Locomotion was assessed by the walkingtrack test at 4, 8, and 12 weeks after transection. Improved recovery of locomotion in etifoxine-treated animals was followed by increase in SFI index, calculated using the de Medinaceli method. SFI, sciatic functional index.
Functional Results
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Therefore, etifoxine could regulate various biological functions in cells. Moreover, TSPO may also affect neurite outgrowth by controlling the rate of neurosteroid formation.16 The neurosteroids PREG, PROG, and DHEA are key molecules in neuronal regeneration. The levels of the key molecules PREG and PROG have been shown to increase in injured sciatic nerves and to produce neurotrophic effects in vitro and in vivo.17 PROG increased neurite outgrowth of DRG explants and promoted regeneration in cryolesioned sciatic nerves and the remyelination of regenerated nerve fibers.17,18 In addition, estradiol (a steroid) leads to increased GDNF expression, which may occur through rapid rises in Ca2þ levels followed by CREB phosphorylation. CREB phosphorylation ultimately leads to increased GDNF expression.19 Further experiment is still needed to test the hypothesis that TSPO activation will lead to an increase of steroidogenesis and that increased steroids may affect gene expression through CREB phosphorylation. Recent studies, however, showed that etifoxine could induce acute hepatitis in human patients. Although a causal relationship with etifoxine administration is supported though the exclusion of other common causes of hepatitis,20 a direct mechanism of action still requires further investigation. Given those side effects, studies involving varied etifoxine concentrations are needed. In our research, we found that PC12 cell neuronal-like outgrowth increased in a dose-dependent manner. However, GDNF expression remained elevated at etifoxine concentrations of 20 µM and less, but when 40 µM etifoxine was administered to PC12 cells, GDNF expression dropped. This result suggests that 40 µM etifoxine may have a reduced therapeutic effect compared with 20 µM etifoxine. Considering our data and the known side effects, we suggest that 20 µM etifoxine may be the optimal concentration for PC12 cell studies. We also assessed the effect of etifoxine in an in vivo model. Histological assessment showed that etifoxine-enhanced nerve fiber growth, and etifoxine treatment produced regenerated nerves that were similar in structure to the regenerated nerves of the autograft group (►Fig. 3). In contrast, myelinated nerve fibers of the ANA group were less dense, uneven in size, and less myelinated. These differences demonstrated that etifoxine could provide therapeutic benefit in a rat model. Etifoxine has been shown to have anti-inflammatory effects by modulating TNF-α mRNA expression in the peripheral nervous system (PNS),8 and other potential inflammatory molecules modulated by etifoxine remain unidentified. Nerve injury initiates an inflammatory response and induces the expression of proinflammatory cytokines such as TNF-α, IL-1β, and Interferon (INF)-γ.21,22 Inflammatory mediators play a role in nerve regeneration, and downregulation of these factors can moderately improve nerve regeneration.23 In this study, etifoxine administration attenuated nerve injury-induced production of proinflammatory cytokines such as TNF-α and IL-1 (►Fig. 5). However, IL-6 (a multipotent cytokine possessing proinflammatory and anti-inflammatory effects) has been shown to be involved in cell proliferation, survival, differentiation, and death.24 Examining our data, etifoxine failed to attenuate the elevation of IL-6 production after transection injury (►Fig. 5). Along with its anti-inflammatory properties,
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etifoxine could potentially enhance in vivo production of neurotrophic factors. In our study, etifoxine treatment increased NGF mRNA GDNF and VEGF mRNA levels. When GDNF was intrathecally delivered after avulsion and reimplantation of the lumbar spinal root, compared with untreated group a greater number of motoneurons not only survived but also were retrograde labeled, which indicated that GDNF facilitated motor axon regeneration into the reimplanted root.25 In addition, VEGF is a potent angiogenic peptide that promotes endothelial cell division and enhances neovascularization. The neurotrophic and neuroprotective effects of VEGF directly influence neuronal behavior.26,27 These findings suggest that etifoxine affects many trophic factors and pathways, thus offering new insights into the design of therapeutic strategies that promote nerve regeneration.
Conclusion In conclusion, this study indicated that optimal concentrations of etifoxine (< 20 µM) provided the most beneficial effects in PC12 cells and that these effects were dose dependent. A higher than optimal etifoxine concentration (40 µM) produced adverse effects on PC12 cells. Moreover, etifoxine greatly enhanced the repair of acellular nerve grafts by selectively modulating inflammatory responses to injury and increasing the expression of neurotrophic factors.
Acknowledgments We are grateful to Yuexiong Yang for technical assistance. This study was supported by grants from National High Technology Research and Development Program of China (contract grant number: 2012AA020507), National Natural Science Foundation of China (30700847, 31070869), Guangdong Natural Science Foundation (9251008901000017), and Guangdong Program in Science and Technology (2010B031100006).
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nerve regeneration in acellular nerve grafts with local release of nerve growth factor. Microsurgery 2009;29(4): 330–336 Girard C, Liu S, Cadepond F, et al. Etifoxine improves peripheral nerve regeneration and functional recovery. Proc Natl Acad Sci U S A 2008;105(51):20505–20510 Girard C, Liu S, Adams D, et al. Axonal regeneration and neuroinflammation: roles for the translocator protein 18 kDa. J Neuroendocrinol 2012;24(1):71–81 Verleye M, Akwa Y, Liere P, et al. The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain. Pharmacol Biochem Behav 2005;82(4): 712–720 Mills C, Makwana M, Wallace A, et al. Ro5-4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats. Eur J Neurosci 2008;27(4):937–946 Zhou X, He X, He B, et al. Etifoxine promotes glial-derived neurotrophic factor-induced neurite outgrowth in PC12 cells. Mol Med Rep 2013;8(1):75–80 Sondell M, Lundborg G, Kanje M. Regeneration of the rat sciatic nerve into allografts made acellular through chemical extraction. Brain Res 1998;795(1-2):44–54 Faul F, Erdfelder E, Buchner A, Lang AG. Statistical power analyses using GPower 3.1: tests for correlation and regression analyses. Behav Res Methods 2009;41(4):1149–1160 Soustiel JF, Zaaroor M, Vlodavsky E, Veenman L, Weizman A, Gavish M. Neuroprotective effect of Ro5-4864 following brain injury. Exp Neurol 2008;214(2):201–208 Mills CD, Bitler JL, Woolf CJ. Role of the peripheral benzodiazepine receptor in sensory neuron regeneration. Mol Cell Neurosci 2005; 30(2):228–237
and myelin formation by Schwann cells. Science 1995;268(5216): 1500–1503 Koenig HL, Gong WH, Pelissier P. Role of progesterone in peripheral nerve repair. Rev Reprod 2000;5(3):189–199 Ivanova T, Karolczak M, Beyer C. Estradiol stimulates GDNF expression in developing hypothalamic neurons. Endocrinology 2002;143(8):3175–3178 Moch C, Rocher F, Lainé P, et al. Etifoxine-induced acute hepatitis: a case series. Clin Res Hepatol Gastroenterol 2012;36(5):e85–e88 Shamash S, Reichert F, Rotshenker S. The cytokine network of Wallerian degeneration: tumor necrosis factor-alpha, interleukin1alpha, and interleukin-1beta. J Neurosci 2002;22(8):3052–3060 Taskinen HS, Olsson T, Bucht A, Khademi M, Svelander L, Röyttä M. Peripheral nerve injury induces endoneurial expression of IFN-gamma, IL-10 and TNF-alpha mRNA. J Neuroimmunol 2000;102(1):17–25 Kato K, Liu H, Kikuchi S, Myers RR, Shubayev VI. Immediate antitumor necrosis factor-alpha (etanercept) therapy enhances axonal regeneration after sciatic nerve crush. J Neurosci Res 2010;88(2): 360–368 Hodge DR, Hurt EM, Farrar WL. The role of IL-6 and STAT3 in inflammation and cancer. Eur J Cancer 2005;41(16):2502–2512 Bergerot A, Shortland PJ, Anand P, Hunt SP, Carlstedt T. Cotreatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury. Exp Neurol 2004; 187(2):359–366 Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts. Brain Res 1999;846(2):219–228 Storkebaum E, Lambrechts D, Dewerchin M, et al. Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat Neurosci 2005;8(1):85–92
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Etifoxine Promotes Glia-Derived Neurite Outgrowth In Vitro and In Vivo Ting Dai, PhD1, Xiang Zhou, PhD, MD2, Yanan Li, PhD3 Bo He, MD, PhD2 Zhaowei Zhu, MD, PhD2 Canbin Zheng, MD, PhD2 Shuang Zhu, MD, PhD2 Qingtang Zhu, MD, PhD2 Xiaolin Liu, MD, PhD2
Medical University, Guangzhou, China 2 Department of Microsurgery and Orthopaedic Trauma, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China 3 Department of Biochemistry & Molecular Biology, School of Basic Science, Guangzhou Medical University, Guangzhou, China,
Address for correspondence Xiaolin Liu, MD, PhD, 58 Zhongshan Er Road, Guangzhou, China (e-mail: [email protected]).
These authors contributed equally to this work. J Reconstr Microsurg 2014;30:381–388.
Abstract
Keywords
► etifoxine ► GDNF ► neurite outgrowth
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 ¼ 0.0046). In vivo studies demonstrated that etifoxine improved sciatic nerve regeneration, modulated immune responses, and boosted neurotrophin expression. Conclusions Because of etifoxine’s 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.
The most common method of repairing injured nerves is to directly suture two severed nerve ends together. However, this method is often impossible if the gap between the two nerve ends is too large to allow for tension-free nerve repair. Several research groups have tried to accelerate nerve regeneration through microsurgical techniques. However, despite recent ad-
vances in these techniques, complete recovery is rarely achieved.1 Acellular nerve allografts (ANAs) derived from native peripheral nerve retain the structure and extracellular matrix (ECM) components of the original nerve and stimulate little host immune response. Previously, our group demonstrated the usefulness of acellular nerve grafts to repair small and large
received October 24, 2013 accepted after revision April 8, 2014 published online June 23, 2014
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DOI http://dx.doi.org/ 10.1055/s-0034-1381751. ISSN 0743-684X.
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1 Department of Biotechnology, School of Basic Science, Guangzhou
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nerve injuries in several animal models.2–4 On the basis of those findings, acellular nerve grafts have been attempted in humans, and a multicenter trial has been completed. Those results indicate that hANA is a safe and effective nerve repair technique for nerve injuries of 1 to 5 cm in size.5 However, because the efficiency and effective repair length of acellular nerve grafts are still low,3,5–7 a promising therapeutic approach could involve combining nerve grafts with techniques to enhance nerve cell regeneration within the graft. Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H3, 1-benzoxazine hydrochloride; Stresam, [Gentilly, Val-deMarne, France], Biocodex) is currently under investigation as a promising therapeutic to promote neuroprotection, accelerate axonal regeneration, and modulate inflammation.8,9 Traditionally, etifoxine is used as an anxiolytic and anticonvulsant drug. Recent research showed that it targets GABA A receptors and the 18-kDa translocator protein (TSPO).10 In addition to etifoxine, Mills et al found that Ro5–4864, another TSPO ligand, significantly enhanced regeneration and functional recovery in the rat peripheral nervous system.11 Zhou et al speculated that TSPO activation will lead to an increase of steroidogenesis and that increased steroids may affect glia-derived neurotrophic factor (GDNF) gene expression through cAMP-response element binding protein (CREB) phosphorylation. Moreover, etifoxine stimulated GDNF-induced neurite outgrowth in PC12 cells,12 but details surrounding optimal therapeutic etifoxine concentrations are still lacking. In this study, we used a well-defined PC12 cell model to test the effects of etifoxine on GDNF-induced neurite outgrowth. Then, we verified its effects using a 10-mm rat sciatic transection model. We found that etifoxine leads to increased neurite outgrowth at an optimal concentration of 20 µM. In addition, in vivo experiments showed that the etifoxinetreated group experienced enhanced nerve fiber growth, and the structure of regenerated nerves was similar to the regenerated nerve structure in the autograft group. These data demonstrate a potential therapeutic role for etifoxine in human patients.
Methods Cell Culture and Neurite Outgrowth Measurements Rat PC12 cells were cultured on collagen-coated plates (5 µg/cm2) in Dulbecco Modified Eagle Medium supplemented with 5% horse serum, 10% FBS, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Etifoxine dissolved in dimethyl sulfoxide (5, 10, 20, and 40 µM final concentration), and then it was added to cultures. Neuronal-like outgrowth was determined after 10 days of treatment using an Olympus IX81 microscope and U-CMAD 3 camera (Olympus, Tokyo, Japan). Cells were counted in six fields of view of the cell culture dish. At least 400 cells were counted per sample, and experiments were repeated at least three times. Cell axon length was analyzed using image analysis software, version 3.2 (SIS, Münster, Germany). PC12 cells with axons longer than the average cell diameter were used for data analysis. The two-tailed Mann–Whitney U-test (GraphPad Prism 4; GraphPad Software, San Diego, CA) was used to Journal of Reconstructive Microsurgery
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assess statistical significance of GDNF release and its bioactivity on PC12 cell axonogenesis.
Real-Time Polymerase Chain Reaction Real-time polymerase chain reaction (RT-PCR) was performed as previously described.12 Total RNA was extracted from 1 106 cells. First strand cDNA was synthesized from total RNA (1 mg) using avian myeloblastosis virus reverse transcriptase (RT) XL primed by 50 pmol of random 9-mers (TaKaRa Biomedicals, Tokyo, Japan). The RT reaction products (10 µL) were utilized as templates in PCR with 0.2 mM of each of the following primers: GDNF, forward 5′-GGTCTACGGAGAGACCGATCCGAGGTGC- 3′ and reverse 5′-TCTCTGGAGCCAGGGTCAGATACATC-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-TGAAGGTCGGTGTCAACGGATTTGGC-3′ and reverse 5′-CATGTAGGCCATGAGGTCCACCAC- 3′. PCR products were separated by electrophoresis with precast 2% agarose gels (Daiichi Pure Chemicals, Tokyo, Japan) and visualized by SYBR Green 2 (FMC BioProducts, Rockland, ME) staining and a UV transilluminator. The PCR product signal intensities were determined with ImageJ, and the amount of GDNF PCR product was determined by normalizing to the GAPDH signal intensity.
Surgical Procedures A total of 50 male Sprague-Dawley rats (range, 200–230 g) were used in this study. In addition, six male Wistar rats were used for nerve grafts. All animal procedures were approved by the Experimental Animal Administration Committee of Sun Yat-sen University, China. Efforts were made to minimize pain and suffering during the postsurgery period. Animals were randomly divided into the following three groups: (1) autograft group (n ¼ 18), where a 10-mm sciatic nerve segment was removed, inverted, and reimplanted into the surgical gap; (2) simple acellular nerve graft group (n ¼ 18), where a 10-mm sciatic nerve segment was replaced by a 12-mm acellular nerve graft, and daily intraperitoneal (i.p.) injections of 0.5 mL/100 g vehicle (1% Tween-80 in 0.9% NaCl solution) were administered; and (3) acellular nerve graft plus etifoxine group (n ¼ 18), where daily i.p. etifoxine injections (Batch 285; Biocodex, Gentilly, France) were administered at a dosage of 50 mg/kg for 7 days. The 50 mg/kg dosage was selected because it had been previously shown to exhibit anxiolytic-like effects in rats.8 Male Sprague-Dawley rats (range, 200–230 g) were deeply anesthetized by intraperitoneal pentobarbital injection (75 mg/ kg), and surgical procedures were performed under deep anesthesia (40 mg/100 g of chloral hydrate) and aseptic conditions. The animals were monitored up to 4 weeks postsurgery. Rats were allowed free access to food and water ad libitum. To prepare nerve grafts, Wistar rats were euthanized by intraperitoneal sodium pentobarbital injection (0.5 mL, 60 mg/mL). The sciatic nerves were excised, cleansed of external debris, and treated with chemical detergents to produced acellular allografts. This method for isolating acellular grafts was derived from the method developed by Sondell et al. 13
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Four weeks post-surgery, rats were euthanized, and tissues were prepared for histological examination. The repaired nerve segments were harvested from all the three groups. Toluidine blue staining was performed as previously described.3 Briefly, nerve segments were harvested and quickly immersed in a 2.5% Na-cacodylate-buffered glutaraldehyde solution for 2 hours and fixed for 2 hours in 2% Na-cacodylatebuffered osmium tetroxide. Then, the nerve segments were serially dehydrated in increasing concentrations of ethanol, infiltrated and embedded in Epon 812 (Ted Pella, CA). A 1 µm cross-section of the nerve segment was obtained and then stained with toluidine blue to evaluate the efficacy of nerve regeneration. Analysis of the average number of myelinated axons and the fiber diameter was performed using an Olympus BX60 (Olympus, Melville, NY, USA) microscope and the Image-Pro-Plus (Version 6.0) quantitative morphology software. For each sample, images were acquired from three random fields, and the number of axons and the nerve fiber diameters were measured. Six samples from each group were tested for statistical significance. Transmission electron microscopy was employed to evaluate myelin sheath regeneration. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with a Philips CM120 (Philips, Eindhoven, Netherlands)
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transmission electron microscope equipped with an image acquisition system (8,000 magnification) to measure myelin sheath thickness. Images from 10 random fields of each ultrathin nerve section were analyzed with the ImagePro Plus software.
Quantitative Real-Time Polymerase Chain Reaction In nerve segments, quantitative real-time PCR was used to detect and measure mRNA levels of nerve growth factor (NGF), GDNF, vascular endothelial growth factor (VEGF), tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). Primers for rat NGF, GDNF, VEGF, TNF-α, IL-1, IL-6, and GAPDH were designed using the Primer Express 2.0 software (Applied Biosystems, Foster City, CA) and purchased from Huirui Bio Technologies (Shanghai, China). One week postsurgery, six rats per group were humanely sacrificed, and their sciatic nerves were stored in RNA-Later (Ambion, Austin, TX) at 20°C until use. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA), purified on RNeasy minicolumns (Qiagen, Valencia, CA), and treated with RNase free DNAse I (Qiagen , Valencia, CA). The RNA purity (OD260/280 absorption ratio) was measured at 1.9 to 2.0. cDNA was synthesized using a SuperScript II first-strand RT-PCR kit (Invitrogen , Carlsbad, CA). Gene expression was measured by qPCR (MX4000; Stratagene, La Jolla, CA) with 50 ng of rat cDNA and TaqMan Fast
Fig. 1 (A) Microscopic view of PC12 cells in saline, (B) 5 µM, (C) 10 µM, (D) 20 µM, and (E) 20 µM Etifoxine, 10 days of cultivation, figures demonstrating significant increase of axonal sprouting activity. (F) Statistics analysis shows that increase of average axon length was a dosagedependent increase model. A two-tailed Mann–Whitney U-test was used for statistical assessment. p < 0.05, p < 0.01.
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Histological Examination
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Universal PCR Master Mix (2) (Applied Biosystems) with a one-step program (50 cycles of 95°C for 10 minutes, 95°C for 30 seconds, and 60°C for 1 minute). For each gene, duplicate samples without cDNA (no template control) demonstrated the lack of contaminating DNA. Gene expression levels were normalized to GAPDH and quantified by using the comparative critical threshold (Ct) method.
Statistical analysis of PC12 cell neuronal-like outgrowth revealed highly significant etifoxine-induced axonogenesis, even when comparing 5 µM etifoxine (p ¼ 0.43; 28.25 3.43 µm) with the saline control (20.22 3.29 µm) (►Fig. 1F). The average axon length increased in a concentration-dependent manner and peaked at 40 µM etifoxine, with an average length of 42.26 9.76 µm.
Walking-Track Analysis
Etifoxine Induces GDNF mRNA Expression
Functional nerve regeneration was assessed by calculating the sciatic functional index (SFI) (de Medinaceli et al 1982). In total, 18 rats had been used to assess walking-track behavior. Briefly, after their hind feet were dipped in dark ink, rats walked down a wooden corridor. SFI was calculated using the following formula:
To address the mechanism of etifoxine-induced neuronal-like outgrowth in PC12 cells, we measured GDNF mRNA expression by RT-PCR using GDNF-specific primers as described earlier. After etifoxine treatment, GDNF mRNA expression levels increased by 1.50-fold (p ¼ 0.035) after 5 µM etifoxine treatment compared with the saline treatment (►Fig. 2). These data support the finding that etifoxine induces neuronal-like outgrowth in PC12 cells. Next, we determined the optimal etifoxine concentration that produced the largest induction of GDNF expression. PC12 cells were treated with increasing concentrations of etifoxine, and GDNF expression increased in a concentration-dependent manner from 5 to 20 µM. However, when 40 µM etifoxine was administered, GDNF expression decreased, but this change was not significant compared with 20 µM (1.97 0.23 and 1.91 0.26, p ¼ 0.95).
In this formula, the print length (PL) was defined as the distance from the heel to the third toe; the toe spread (TS) was defined as the distance from the first to fifth toe; and the intermediary toe spread (IT) was defined as the distance from the second to the fourth toe. All three measurements were made on the experimental (E) and normal (N) sides. The recovery of walking function is a gradual process after the surgery. To see the changes along this process, the walking functional test was performed every 4 weeks, beginning on the 4th week and ending on the 12th week.
Histological Examination After examining toluidine blue-stained nerve segments at 8 weeks postsurgery, etifoxine treatment enhanced axon
Statistical Analysis All the results were subjected to statistical analysis using SPSS 11.5 software (SPSS, San Francisco, CA) for Windows. For quantification in the article, two researchers read the results independently, final results come from the mean of those two researchers. Comparisons between two groups were performed by Student t-test. Statistically significant values were defined as p < 0.05. Statistically power was calculated using the software GPower 3.14 Power calculated for the cell study is 0.72. Power calculated for the animal study is 0.28.
Results Etifoxine induces neuronal-like outgrowth in PC12 cells in a dose-dependent manner. To examine whether etifoxine can promote GDNF-induced neurite outgrowth in PC12 cells, the length of outgrown fibers was measured. ►Fig. 1 clearly demonstrates the biological activity of etifoxine, and in agreement with previous reports showing etifoxine-induced neurite extension, a dramatic increase in neuronal-like outgrowth was observed 10 days after etifoxine application (►Fig. 1B). Next, to determine the optimal etifoxine concentration, cultured PC12 cells were treated with increasing concentrations of etifoxine (from 5 to 40 µM). In contrast to 5 µM etifoxine, higher etifoxine concentrations only increased PC12 cell neuronal-like processes after 10 days of treatment. Journal of Reconstructive Microsurgery
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Fig. 2 Expression of GDNF mRNA in drug-treated PC12 cells. The results of real time polymerase chain reaction analysis of GDNF mRNA in PC12 cells. (S, cell treated with saline. 5, cell treated with 5 µm Etifoxine. 10, cell treated with 10 µm Etifoxine. 20, cell treated with 20 µm Etifoxine. 40, cell treated with 40 µm Etifoxine.) A representative result is shown, and the data are presented as the mean and standard deviation of PC12 cell cultures in media (n ¼ 8). p < 0.05, p < 0.01, compared ANAþE with ANA. #p < 0.05, compared 20 µm with 40 µm, determined by two-tailed Student t-test. GDNF, glia-derived neurotrophic factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
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Fig. 3 Histological sections of regenerated nerves. Semithin cross-sections of the distal portion of each nerve graft were stained with toluidine blue 4 weeks postsurgery. (A) Intact rat sciatic nerve. (B) Autograft group. (C) ANA group. (D) Etifoxine group. (E) Average diameter of the nerve. (F) Number of myelinated axons. Groups with etifoxine demonstrated more organized neural architecture, closely approximating the autograft, in comparison to the ANA alone group. Error bars indicate mean standard error of mean Significance of differences was determined by t-test (n ¼ 6). p < 0.05 compared with the etifoxine (ANA þ E) and autologous (auto) groups. ANA, acellular nerve allografts.
regeneration (►Fig. 3). Myelinated fibers were similar in size and shape and symmetrically arranged in the autograft group, but they were randomly spaced and less symmetrical in the etifoxine and ANA groups. The number and diameter of regenerated axons were larger in the etifoxine group than in the autograft group. The total number of axons was similar in the etifoxine and autograft groups, which suggests that axon regeneration was achieved in both the groups (49,537 2,123 vs. 57,523 2,023; p ¼ 0.12). The number of myelinated (toluidine blue positive) axons was significantly increased in the etifoxine group compared with the ANA group (39,105 2,387 vs. 57,523 2,023; p ¼ 0.031) (►Fig. 3E). Although similar in both the etifoxine and autograft groups (2.95 0.17 vs. 2.76 0.21), the average diameter of myelinated fibers was significantly larger in the etifoxine group compared with the ANA group (1.94 0.23 vs. 2.76 0.21; p ¼ 0.025) (►Fig. 3F). Along with histological evidence, transmission electron microscopy revealed regenerated myelinated fibers in the
mid portion of the implant 4 weeks after graft implantation in each group. The myelinated fibers were more compact and uniform in the etifoxine and autograft groups than the fibers of the ANA group. The average myelin sheath thickness of the etifoxine group was larger than the ANA group sheath thickness (1.07 0.17 vs. 0.65 0.13, p ¼ 0.028), but the average myelin sheath thickness was similar in the etifoxine and autograft groups (►Fig. 4D).
Gene Expression RT-PCR was utilized to investigate the potential changes in proinflammatory signals in response to peripheral nerve injury and etifoxine administration. The TNF-α transcript (1.11 0.14 vs. 0.64 0.13, p ¼ 0.034; ►Fig. 5) and IL-1 transcript (1.08 0.18 vs. 0.48 0.11, p ¼ 0.025) levels were reduced in the etifoxine group compared with the control group. Although etifoxine influences the immune response in rat sciatic nerve, it is unknown whether etifoxine influences neurotrophin expression in peripheral nerves. To determine Journal of Reconstructive Microsurgery
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Etifoxine Promotes Neurite Outgrowth
Etifoxine Promotes Neurite Outgrowth
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Fig. 4 Transmission electron micrographs. (A) Autograft group. (B) Acellular nerve allografts group. (C) Etifoxine group. (D) Myelin sheath thickness of each group. Error bars indicate mean standard error of mean Significance of difference was determined by t-test (n ¼ 6). p < 0.05.
whether etifoxine modulates neurotrophin expression after peripheral nerve injury, the expression levels of NGF, BDNF, and VEGF were quantified in regenerating sciatic nerves (►Fig. 5). At 7 days postinjury, etifoxine treatment had a marked neurotrophic effect (NGF, a 1.9-fold increase over control, p ¼ 0.019; GDNF, a 2.1-fold increase over control, p ¼ 0.0036; and VEGF, a 1.7-fold increase over control, p ¼ 0.072).
12 weeks postoperatively, functional recovery was almost the same in the etifoxine and autologous groups (►Fig. 6).
Discussion
Walking track analysis is a useful technique for evaluating motor functional recovery after sciatic nerve injury. The increase in sciatic nerve functional index after treatment indicates peripheral nerve regeneration. As shown in ►Fig. 6, SFI was significantly higher in the etifoxine group than the ANA group 8 weeks postoperatively. However, it is still lower than the autologous group. This result shows that etifoxine treatment enhances functional recovery in ANAs. By
In this study, we found that etifoxine-induced PC12 cell neuronal-like outgrowth, which increased up to 3.27-fold after 10 days and resulted in numerous partially arborescent axons. In addition, the optimal etifoxine concentration was 20 µM, which induced maximal stimulation of GDNF expression and neuronal-like outgrowth. In vivo experiments demonstrated that etifoxine-enhanced peripheral nerve regeneration across 10-mm nerve gaps repaired by acellular nerve grafts. Etifoxine is a TSPO ligand that associates with the mitochondrial permeability transition pore, which allows the electron transport chain to create the transmembrane electrochemical gradient (ΔΨm) driving ATP synthesis.15
Fig. 5 Effects of etifoxine on mRNA expression. mRNA levels in nerve were normalized to GAPDH mRNA levels. Data are expressed as the mean standard error of mean. Significance of difference was determined by t-test (n ¼ 6). p < 0.05, p < 0.01. ANA, acellular nerve allografts; GDNF, glia-derived neurotrophic factor; IL, interleukin; NGF, nerve growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
Fig. 6 Etifoxine treatment improved the functional recovery of transected sciatic nerve. Locomotion was assessed by the walkingtrack test at 4, 8, and 12 weeks after transection. Improved recovery of locomotion in etifoxine-treated animals was followed by increase in SFI index, calculated using the de Medinaceli method. SFI, sciatic functional index.
Functional Results
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Therefore, etifoxine could regulate various biological functions in cells. Moreover, TSPO may also affect neurite outgrowth by controlling the rate of neurosteroid formation.16 The neurosteroids PREG, PROG, and DHEA are key molecules in neuronal regeneration. The levels of the key molecules PREG and PROG have been shown to increase in injured sciatic nerves and to produce neurotrophic effects in vitro and in vivo.17 PROG increased neurite outgrowth of DRG explants and promoted regeneration in cryolesioned sciatic nerves and the remyelination of regenerated nerve fibers.17,18 In addition, estradiol (a steroid) leads to increased GDNF expression, which may occur through rapid rises in Ca2þ levels followed by CREB phosphorylation. CREB phosphorylation ultimately leads to increased GDNF expression.19 Further experiment is still needed to test the hypothesis that TSPO activation will lead to an increase of steroidogenesis and that increased steroids may affect gene expression through CREB phosphorylation. Recent studies, however, showed that etifoxine could induce acute hepatitis in human patients. Although a causal relationship with etifoxine administration is supported though the exclusion of other common causes of hepatitis,20 a direct mechanism of action still requires further investigation. Given those side effects, studies involving varied etifoxine concentrations are needed. In our research, we found that PC12 cell neuronal-like outgrowth increased in a dose-dependent manner. However, GDNF expression remained elevated at etifoxine concentrations of 20 µM and less, but when 40 µM etifoxine was administered to PC12 cells, GDNF expression dropped. This result suggests that 40 µM etifoxine may have a reduced therapeutic effect compared with 20 µM etifoxine. Considering our data and the known side effects, we suggest that 20 µM etifoxine may be the optimal concentration for PC12 cell studies. We also assessed the effect of etifoxine in an in vivo model. Histological assessment showed that etifoxine-enhanced nerve fiber growth, and etifoxine treatment produced regenerated nerves that were similar in structure to the regenerated nerves of the autograft group (►Fig. 3). In contrast, myelinated nerve fibers of the ANA group were less dense, uneven in size, and less myelinated. These differences demonstrated that etifoxine could provide therapeutic benefit in a rat model. Etifoxine has been shown to have anti-inflammatory effects by modulating TNF-α mRNA expression in the peripheral nervous system (PNS),8 and other potential inflammatory molecules modulated by etifoxine remain unidentified. Nerve injury initiates an inflammatory response and induces the expression of proinflammatory cytokines such as TNF-α, IL-1β, and Interferon (INF)-γ.21,22 Inflammatory mediators play a role in nerve regeneration, and downregulation of these factors can moderately improve nerve regeneration.23 In this study, etifoxine administration attenuated nerve injury-induced production of proinflammatory cytokines such as TNF-α and IL-1 (►Fig. 5). However, IL-6 (a multipotent cytokine possessing proinflammatory and anti-inflammatory effects) has been shown to be involved in cell proliferation, survival, differentiation, and death.24 Examining our data, etifoxine failed to attenuate the elevation of IL-6 production after transection injury (►Fig. 5). Along with its anti-inflammatory properties,
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etifoxine could potentially enhance in vivo production of neurotrophic factors. In our study, etifoxine treatment increased NGF mRNA GDNF and VEGF mRNA levels. When GDNF was intrathecally delivered after avulsion and reimplantation of the lumbar spinal root, compared with untreated group a greater number of motoneurons not only survived but also were retrograde labeled, which indicated that GDNF facilitated motor axon regeneration into the reimplanted root.25 In addition, VEGF is a potent angiogenic peptide that promotes endothelial cell division and enhances neovascularization. The neurotrophic and neuroprotective effects of VEGF directly influence neuronal behavior.26,27 These findings suggest that etifoxine affects many trophic factors and pathways, thus offering new insights into the design of therapeutic strategies that promote nerve regeneration.
Conclusion In conclusion, this study indicated that optimal concentrations of etifoxine (< 20 µM) provided the most beneficial effects in PC12 cells and that these effects were dose dependent. A higher than optimal etifoxine concentration (40 µM) produced adverse effects on PC12 cells. Moreover, etifoxine greatly enhanced the repair of acellular nerve grafts by selectively modulating inflammatory responses to injury and increasing the expression of neurotrophic factors.
Acknowledgments We are grateful to Yuexiong Yang for technical assistance. This study was supported by grants from National High Technology Research and Development Program of China (contract grant number: 2012AA020507), National Natural Science Foundation of China (30700847, 31070869), Guangdong Natural Science Foundation (9251008901000017), and Guangdong Program in Science and Technology (2010B031100006).
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