ETIFOXINE PROVIDES BENEFITS IN NERVE REPAIR WITH ACELLULAR NERVE GRAFTS XIANG ZHOU, MD, PhD,1 BO HE, MD, PhD,1 ZHAOWEI ZHU, MD, PhD,1 XINHUA HE, PhD,2 CANBIN ZHENG, MD, PhD,1 JIAN XU, MD, PhD,3 LI JIANG, PhD,1 LIQIANG GU, MD, PhD,1 JIAKAI ZHU, MD, PhD,1 QINGTANG ZHU, MD, PhD,1 and XIAOLIN LIU, MD, PhD1 1
Department of Microsurgery and Orthopedic Trauma, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China 2 Department of Physiology, Medical College of Shangtou University, Shantou, China 3 Department of Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Accepted 19 November 2013 ABSTRACT: 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 SpragueDawley 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
Traumatic injury to peripheral nerves may result in considerable loss of sensory and motor function and thereby in decreased quality of life. Several research groups have tried to accelerate regeneration of traumatized nerves through application of microsurgical techniques. However, despite advancements in these techniques, complete recovery is achieved rarely.1,2 Acellular nerve allografts (ANAs) derived from native peripheral nerve retain Abbreviations: ANA, acellular nerve allograft; CMAP, compound muscle action potential; CNS, central nervous system; DRG, dorsal root ganglia; ECM, extracellular matrix; GF, growth factor; GDNF, glia-derived growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin; NCV, nerve conduction velocity; NGF, nerve growth factor; NF-200, neurofilament-200; PCR, polymerase chain reaction; PB, phosphate buffer; SFI, sciatic functional index; TSPO, translocator protein (18 kDa); VEGF, vascular endothelial growth factor Key words: acellular nerve grafts; etifoxine; nerve regeneration; neurotrophins expression; TSPO This study was supported by grants from National High Technology Research and Development Program of China (2012AA020507), the National Nature Science Grant of China (30700847), 985 program of Sun Yat-sen University (Grant No. 90035-3283312); Specialized Research Fund for the Doctral Program of Higher Education (SRFDP) (Grant No. 20120171120075); and the Doctoral Start-up Project of the Guangdong Nature Science Foundation (S201204006336). The first 2 authors (X.Z. and B.H.) contributed equally to this article. Correspondence to: X. Liu; e-mail: [email protected] C 2013 Wiley Periodicals, Inc. V
Published online 24 November 2013 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24131
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the structure and extracellular matrix (ECM) components of the original nerve and stimulate only a low host immune response. In previous studies, our group demonstrated the usefulness of acellular nerve grafts for repair of small and large defects in many animal models of peripheral nerve injury.3–5 Based on those findings, acellular nerve grafts have been attempted in humans, and a multicenter trial has shown good results (unpublished results). However, some problems remain. The efficiency of nerve cell regeneration in ANAs is low,3,6,7 so we tried to combine grafting with ways to enhance nerve cell regeneration in the graft. Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3, 1-benzoxazine hydrochloride; Stresam, iocodex) is currently under investigation as a therapeutic way to promote neuroprotection, accelerate axonal regeneration, and modulate inflammation.8,9 It is used traditionally as an anxiolytic and anticonvulsant drug. Recent research has shown that etifoxine targets GABA-A receptors and the 18kDa translocator protein TSPO.10,11 TSPO is multifunctional and is localized mainly in the outer mitochondrial membrane.12 In rats, ligand binding stimulates the cholesterol transfer function of TSPO. Campioli et al.13 found that expression of TSPO 18 kDa increases after a stressful stimulus. Overexpression of TSPO increases the motility, transmigration, and proliferation properties of C6 rat glioma cells.14 After peripheral nerve injury, TSPO expression is increased transiently in many cells such as dorsal root ganglia (DRG) neurons, Schwann cells, and macrophages.15,16 Also, TSPO ligands have been shown to exert neuroprotective effects and reduce neural inflammation in the central nervous system (CNS),17,18 protect motoneurons from degeneration after facial nerve injury, reduce sensorimotor deficits after acrylamide intoxication, prevent aging-associated peripheral nerve degeneration,19,20 and stimulate outgrowth of DRG neurites.21 Mills et al. found that the TSPO ligand Ro5–4864 significantly enhances regeneration and functional recovery in the peripheral nervous system of rats.22 Girard et al. found that etifoxine can enhance the repair of peripheral nerve damage MUSCLE & NERVE
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(i.e., accelerate axonal regrowth and improve macrophage responses) in rats.8 All of these experimental observations strongly suggest that TSPO ligands may be used to stimulate axonal regeneration and modulate macrophage activation in damaged peripheral nerves. However, the role of etifoxine in peripheral nerve regeneration and functional recovery after acellular nerve allograft implantation is unknown. Also unknown is whether etifoxine exerts its effect through pathways other than the immune response pathway. We now report that adding etifoxine to the acellular nerve allograft procedure for treatment of peripheral nerve injury improves axonal regrowth and immune responses. Moreover, its administration not only increases the rate but also the extent of functional recovery from sciatic nerve injury. METHODS Surgical Procedures. All animal procedures were approved by the experimental animal administration committee of Sun Yat Sen University. Efforts were made to minimize the suffering of animals during the postsurgery period. Male Sprague-Dawley rats (200–230 g) were anesthetized deeply by intraperitoneal injection of pentobarbital (75 mg/kg). Under aseptic conditions, the skin of the left leg was cut parallel to the femur. The sciatic nerve was exposed by splitting the superficial gluteus muscle. 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. Nerve grafts were sutured microsurgically to the proximal and distal nerve stumps using a 10-0 nylon suture (Sharpoint; Surgical Specialties Corp., Reading, Pennsylvania), with suture of the epineurial sheathes of the sciatic nerve. The rats were divided randomly into 3 groups: (1) an autograft group (n 5 24), in which a 15-mm segment of the sciatic nerve was removed, inverted, and reimplanted into the gap; (2) a simple acellular nerve graft group (n 5 24), in which a 15-mm segment of the sciatic nerve was replaced with an 18-mm acellular nerve graft, with daily intraperitoneal injections of 0.5 ml per 100 g vehicle (1% Tween-80 in 0.9% NaCl solution); and (3) an acellular nerve graft plus etifoxine group (n 5 24), in which the 15-mm segment of the sciatic nerve was replaced with an 18-mm acellular nerve graft, with daily intraperitoneal injections of etifoxine (Batch 285; Biocodex, Gentilly, France) at a dose of 50 mg/kg for 7 days. Etifoxine has been shown to exhibit effects in rats.8 The overlying muscle and skin of the thigh were sutured with 6-0 and 4-0 polyamide sutures. Treatment was started 24 hours after surgery. The animals were allowed to survive up to 12 weeks after surgery. 236
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For nerve graft preparation Wistar rats were killed by intraperitoneal injection with sodium pentobarbital (0.5 ml, 60 mg/ml). The sciatic nerves were excised, cleaned of external debris, and treated with chemical detergents to produce acellular allografts. The method for isolating acellular grafts was based on a procedure developed by Sondell et al.23 Immunohistochemistry. Two weeks after surgery, 6 rats per group were anesthetized with pentobarbital (1.5 g/kg, intraperitoneally) and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at a pH of 7.2–7.4 at 4 C. Rat sciatic nerves were excised, postfixed in 4% paraformaldehyde for 3 hours, dipped in 30% sucrose (in 0.1 M PB) overnight, sectioned (16 lm thickness) in the longitudinal direction on a freezing microtome (Leica CM3050S; Leica, Wetzlar, Germany), and collected into 0.01 M phosphate buffered saline. Immunohistochemical studies were performed using a monoclonal anti–neurofilament 200 (NF200) antibody (Sigma-Aldrich, Tokyo, Japan). The frozen sections were preincubated in 3% hydrogen peroxide and 10% normal rabbit serum for 10 minutes to block nonspecific binding of immunoglobulin, incubated at room temperature overnight with monoclonal NF-200 antibodies (diluted 1:400 in PB), washed 3 times with 0.01 M phosphatebuffered saline (PBS), incubated with Cy3conjugated donkey anti-rabbit IgG (1:300; Jackson ImmunoResearch, West Grove, Pennsylvania), rinsed 3 times with 0.01 M PBS, mounted on gelatin-coated slides, and air-dried. The images of the stained sections were captured with a fluorescence microscope attached to a CCD spot camera (DFC350FX/DMIRB; Leica) and processed with Leica IM50 software. Histological Examination. Four weeks after surgery, 6 rats per group were euthanized for histological examination. The gastrocnemius muscles and repaired nerve segments were harvested in all 3 groups. The muscle samples were postfixed with formalin, embedded in paraffin, and sectioned transversely. The sections were deparaffinized, rehydrated in an alcohol series, and stained with hematoxylin and eosin. For each sample, photographs were taken from 3 random fields and analyzed using ImagePro Plus (version 6.0) software to measure the area of the muscle fibers. Six samples from each group were used for statistical analysis. Toluidine blue staining was performed as described previously.3 Briefly, nerve graft segments were harvested and quickly immersed in 2.5% Nacacodylate–buffered glutaraldehyde solution for 2 MUSCLE & NERVE
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h, fixed for 2 h in 2% Na-cacodylate–buffered osmium tetroxide, serially dehydrated in increasing concentrations of ethanol, infiltrated and embedded in Epon 812 (Ted Pella, Redding, California), sectioned (1 lm thickness), and stained with toluidine blue to evaluate the efficacy of nerve regeneration. Transection was performed in the middle of the nerve graft segments. Analysis of the average number of myelinated axons and fiber diameter was performed using an Olympus BX60 microscope and ImagePro Plus (version 6.0) quantitative morphology software. For each sample, photographs were taken from 3 random fields and analyzed to measure the number and diameter. Six samples from each group were used for statistical analysis. Transmission electron microscopy was employed to evaluate myelin sheath regeneration. Ultrathin sections were stained with lead citrate and uranyl acetate and examined under a Philips CM120 transmission electron microscope equipped with an image acquisition system at 80003 magnification for measuring thickness of myelin sheaths. Photographs from 10 random fields of each ultrathin nerve section were analyzed with ImagePro Plus software. Walking-Track Analysis. Functional nerve regeneration was assessed by calculating the sciatic functional index (SFI).24 Briefly, after the hind feet were dipped in dark ink, rats walked down a wooden corridor. SFI was calculated using the following formula: EPL1NPL ETS1NTS 1 109:53 SFI 5 238:33 NPL NTS EIT 1NIT 28:8 1 13:33 NIT
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 toes; and the intermediary toe spread (IT) was defined as the distance from the second to fourth toes. All 3 measurements were made on the experimental (E) and normal (N) sides. The walking functional test was performed every 2 weeks, beginning at week 2 and ending at week 12. Electrophysiological Testing. Twelve weeks after surgery, 6 rats per group were anesthetized, placed in the prone position for better exposure of the left sciatic nerve, and tested by placement of stimulating crook-shaped silver needle electrodes on the proximal and distal ends of the grafts. Normal compound muscle action potentials (CMAPs) on the contralateral side were also recorded for comEtifoxine in Nerve Regeneration
parison. A personal computer was used to set the conditions, including the frequency and amplitude of the stimulation signal, and recordings were performed using a Nicolet Viking electrodiagnostic system (Nicolet Instrument Corp., Madison, Wisconsin). Digitized data were stored on computer. The peak amplitude and nerve conduction velocity (NCV) were calculated. Real-Time
Quantitative
Polymerase
Chain
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA levels of nerve growth factor (NGF), glia-derived growth factor (GDNF), and vascular endothelial growth factor (VEGF) in the nerve segment. To measure the effect of etifoxine, the etifoxine and saline groups were used in this assay. Primers for rat NGF, GDNF, VEGF, and glyceraldehyde 3phosphate dehydrogenase (GAPDH) were designed using Primer Express v2.0 software (Applied Biosystems, Foster City, California) and obtained at Huirui Bio Technologies (Shanghai, China). One week after surgery, 6 rats per group were euthanized, and their sciatic nerves were stored in RNA-Later (Ambion, Austin, Texas) at 220 C. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, California), purified on RNeasy minicolumns (Qiagen, Valencia, California), and treated with RNase-free DNAse I (Qiagen). The RNA purity (OD260/280 absorption ratio) was determined to be about 1.9–2.0. cDNA was synthesized using a first-strand RT-PCR kit (SuperScript II; Invitrogen). Gene expression was measured by qPCR (MX4000; Stratagene, La Jolla, California) with 50 ng of rat cDNA and 2 3 TaqMan universal PCR master mix (Applied Biosystems) with a 1-step program (95 C for 10 minutes, 95 C for 30 seconds, and 60 C for 1 minute for 50 cycles). Duplicate samples without cDNA (no-template control) for each gene showed no contaminating DNA. Gene expression levels were normalized to GAPDH and were quantified using the comparative critical threshold (Ct) method. Reaction.
Statistical Analysis. All numerical data are given as mean 6 standard error (SE). All results were subjected to statistical analysis using SPSS v11.5 software for Windows (student version). Statistically significant values were defined as P < 0.05. RESULTS Density of Regenerated Axons.
Animals (n 5 6 per group) were euthanized to measure neurofilament protein expression 2 weeks after surgery. Evidence indicates that increased expression of neurofilament proteins reflects early regenerative potential.25 Analysis of immunostained neurofilaments (Fig. 1) demonstrated that expression of MUSCLE & NERVE
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FIGURE 1. Immunohistochemistry analysis of longitudinal sections in the middle of the bridge 2 weeks after surgery. Repair using (A) autologous sciatic nerve, (B) ANA alone, and (C) ANA combined with etifoxine treatment. (D) Neurofilament expression was greater in the autologous group than in the etifoxine group and greater in the etifoxine group than in the ANA group. **P < 0.01 and ***P < 0.001, determined by 2-tailed Student t-tests.
neurofilament proteins was enhanced significantly by etifoxine treatment compared with nontreatment (546,953 6 22,110 vs. 455,760 6 24,979, P < 0.01), but remained significantly lower than expression in the autografts (P < 0.001). Histological Examination. Hematoxylin–eosin (H&E) staining was performed to assess the atrophy of rat gastrocnemius muscle resulting from sciatic nerve damage. The end of the atrophic process is accompanied by gradual recovery of sciatic nerve function.26 The contralateral gastrocnemius with intact nerve (the positive control) had a normal histological appearance with no muscle atrophy (Fig. 2). In contrast, the ipsilateral gastrocnemius muscle was atrophied. The average percentage of muscle fiber area was significantly higher after etifoxine treatment than saline treatment 4 weeks postoperatively (54.1 6 2.3% vs. 66.5 6 1.8%, P < 0.01), confirming a better functional recovery after etifoxine. Examination of the toluidine blue–stained nerve segments at the mid-portion in 3 experimental groups at 8 weeks showed that adding etifoxine greatly enhanced axon regeneration (Fig. 3). Myelinated fibers were of similar size and shape 238
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and were arranged symmetrically in the autograft group, but they were spaced randomly and were less symmetrically shaped in the etifoxine and ANA groups. The number of regenerated axons and axon diameter were larger in the etifoxine group. The number of axons was similar in the etifoxine and autograft groups, suggesting regeneration was achieved in both groups (49,533 6 1245 vs. 54,674 6 2486, P > 0.05). The number of myelinated (toluidine blue positive) axons was significantly higher in the etifoxine group than in the ANA group (40,523 6 1436 vs. 49,533 6 1245, P < 0.001) (Fig. 4A). Although similar in the etifoxine and autograft groups (2.86 6 0.15 vs. 2.69 6 0.21), the average diameter of myelinated fibers was significantly larger in the etifoxine group than in the ANA group (1.93 6 0.33 vs. 2.69 6 0.21, P < 0.05) (Fig. 4B). Transmission electron microscopy revealed regeneration of myelinated fibers in the midportion of the implant 1 month after implantation in each group. The myelinated fibers were more compact and uniform in the etifoxine and autograft groups than in the ANA group. The myelin MUSCLE & NERVE
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FIGURE 2. H&E staining of gastrocnemius muscle. (A) Positive control. (B) Autograft group. (C) ANA group. (D) Etifoxine group.
sheath thickness was greater in the etifoxine group than in the ANA group (1.02 6 0.13 vs. 0.66 6 0.10, P < 0.05), but was similar in the etifoxine and autograft groups (Fig. 5D). Walking-track analysis is a useful technique for evaluating motor functional recovery after sciatic nerve injury. The increase in sciatic nerve functional index (SFI) after treatment indicates peripheral nerve regeneration. As shown in Figure 6, the level of regeneration was significantly higher in the etifoxine group than in the ANA group 8 weeks postoperatively, but still lower than in the autologous group. This result shows that etifoxine treatment enhances functional recovery in ANAs. By 10 weeks postoperatively, functional recovery was almost the same in the etifoxine and autologous groups (Fig. 6).
Functional Results.
Improvement in Electrophysiology. Compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were used to evaluate electrophysiological recovery after surgery. The change in CMAP (% CMAP) was significantly higher in the autograft group (49.6%) than in the ANA (32.3%) and etofoxine (37.8%) groups, but was similar in the ANA and etifoxine groups (Fig. 7A). The Etifoxine in Nerve Regeneration
change in NCV (% NCV) was significantly higher in the autograft group (84.1%) than in the ANA group (56.7%) and significantly higher in the etifoxine group (77.4%) than in the ANA group, but it was similar in the etifoxine and autograft groups (Fig. 7B). These results suggest that etifoxine enhances axonal regeneration. Although etifoxine influences the immune response in rat sciatic nerve, it is unknown whether it influences neurotrophic expression in peripheral nerves. To determine whether etifoxine modulates neurotrophic expression after peripheral nerve injury, the expression of NGF, BDNF, and VEGF was quantified in the regenerating sciatic nerve (Fig. 8). At 7 days after injury, etifoxine treatment had a marked neurotrophic effect (NGF: 1.9-fold increase over control, P < 0.05; GDNF: 2.0-fold increase over control, P < 0.01; VEGF: 1.7-fold increase over control, P < 0.01).
Gene Expression.
DISCUSSION
Girard et al.8 combined etifoxine treatment with silicone tube implantation to repair injured rat sciatic nerve. Silicone or other materials have been used to construct nerve guides for this MUSCLE & NERVE
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FIGURE 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. Thin (1 lm) sections of sciatic nerve specimens were stained with 1% toluidine blue for qualitative examination of the midline of the graft by light microscopy. Groups treated with etifoxine demonstrated more organized neural architecture, closely approximating the autograft, in comparison to the ANA alone group.
purpose.27–29 Nerve graft success depends on various factors: (1) the wall thickness and structural integrity of the graft should be retained to prevent collapse; (2) the materials used to support the graft should be biodegradable and not cytotoxic and provide a suitable matrix for cell adhesion and cell migration; and (3) the grafts should be immunologically tolerated by recipients. ANAs derived from native peripheral nerves retain the
structure and ECM components of the original tissue and stimulate only weak host immune responses. Once implanted, these acellular nerves serve as a natural scaffold into which surrounding cells readily migrate, forming the foundation for new tissue. Thus, acellular nerve implantation is a more promising strategy than other nerve implantation techniques.30 We also believe etifoxine treatment is a good way to improve ANA outcome.
FIGURE 4. (Left) Average diameter of the nerve. (Right) Number of myelinated axons. Error bars indicate mean 6 SEM. Significance of differences was determined by t-test. Results were obtained from 6 rats in each group. *P < 0.05 and **P < 0.001 compared with the etifoxine (ANA1E) and autologous (Auto) groups, determined by 2-tailed Student t-tests. 240
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FIGURE 5. Transmission electron micrographs. (A) Autograft group. (B) ANA group. (C) Etifoxine group. (D) Myelin sheath thickness of each group. Error bars indicate mean 6 SEM. Significance of difference was determined by t-test. *P < 0.05.
Previous reports have shown that muscles may atrophy irreversibly after nerve damage and lose function before axonal regeneration into distal targets. Thus, the speed of regeneration is important in peripheral nerve regeneration. Our results show that in the early stage of nerve regeneration etifoxine treatment accelerated nerve regeneration in ANA. Regeneration of peripheral axons to reach the muscle end-plates weeks or months before atrophy can be very beneficial. In our study, atrophy of gastrocnemius muscles was less pronounced when etifoxine treatment was provided after acellular graft implantation. Histological assessment showed that nerve fibers grew better in the etifoxine group, and the structure of regenerated nerves was similar to that in the autograft group (Fig. 3). In contrast, myelinated nerve fibers in the ANA group were less dense, of uneven size, and less myelinated. H&E stains revealed that gastrocnemius muscle atrophy 3 months after implantation was most obvious in the ANA group with the least percentage of muscle fiber area, suggesting little recovery of nerve function. However, the recovery level in rats treated with etifoxine approached that of the autologous group. In the third part of our study, walking-track analysis was conducted to evaluate function. When measuring experimental nerve recovery, selection of the appropriate methods of evaluation is crucial. In 1982, de Medinaceli et al. first reported that the SFI could be used to evaluate total lower limb Etifoxine in Nerve Regeneration
function, including nerve, muscle, and joint function in rats.24 Walking-track analysis is a noninvasive method to assess sciatic nerve function based on measurement of footprints.23 Because proper walking demands coordinated functions, such as sensory input, motor control, and cortical integration, SFI may be a better measure of function than electrophysiological and histomorphometric measures of axon growth, especially when the research focus is on functional outcome.31 In our study, walking-track analysis findings paralleled histologic findings. Early recovery of SFI is hard to achieve, although histological analyses show good results in
FIGURE 6. Etifoxine treatment improved the functional recovery of transected sciatic nerve. Locomotion was assessed by the walking-track test, and footprints were monitored at 2, 4, 6, 8, 10, and 12 weeks after transection. Improved recovery of locomotion in etifoxine-treated animals was followed by increase in sciatic functional index (SFI), calculated using the de Medinaceli method.23 *P < 0.05 and **P < 0.01, comparing ANA1E with ANA; and ###P < 0.001, comparing ANA1E with Auto, determined by 2-tailed Student t-tests. MUSCLE & NERVE
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FIGURE 7. Electrophysiological evaluation. Nerve conduction velocity (NCV) and compound muscle action potentials (CMAPs) were measured at 12 weeks after sciatic nerve transection. The ratio of nerve conduction latency (B) and CMAP (A) on the injured side compared with the normal side was calculated 12 weeks after injury. *P < 0.05 (n 5 6).
2 weeks, because the recovery of function needs more than just histological recovery. However, improvement was significantly better in the etifoxine and autologous groups than in the ANA group after 8 weeks, but it was not significantly different between the etifoxine and autologous groups, again showing the effectiveness of etifoxine treatment. Electrophysiological analysis is a useful test of functional recovery after nerve injury.32,33 CMAP peak amplitude and NCV decrease after nerve injury.34,35 Electrophysiological recordings demonstrated almost the same level of improvement in nerve conduction velocity in the etifoxine and autologous groups. However, CMAP amplitude was similar in the etifoxine and ANA groups (Fig. 7). Studies suggested that the CMAP amplitude reflects the number of axons reinnervating the muscle and is related to the amount of acetylcholine release, and nerve conduction latency is related inversely to motor function improvement.36,37 Thus, the effect of etifoxine on motor function improvement may be more profound than its effect on acetylcholine release. The relatively small sample size may also account for this result. Etifoxine may also enhance synthesis of neurotrophic factors. Neurotrophic factors are a category of polypeptides or proteins that are released by neurons or non-neuronal cells and have an extensive impact on both the central and peripheral nervous systems.38 In our study, the amount of NGF mRNA was increased after etifoxine treatment. Exogenously applied NGF has been reported to prevent the death of axotomized sensory neurons.39,40 However, the stability of NGF is limited under physiological conditions41,42 and may be reduced further by exposure to organic solvents, shearing, and acidic degradation. This may explain why extrinsic NGF is effective. In our study, NGF was increased endogenously to better promote regeneration. In addition to NGF mRNA, GDNF and VEGF mRNA were also found to increase after etifoxine treatment. GDNF purified from the glial cell line B49 was shown to protect dopaminergic neurons from the rat embryonic midbrain.43 When GDNF 242
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was delivered intrathecally after avulsion and reimplantation of lumbar spinal roots, more motoneurons not only survived but were labeled retrogradely, indicating that GDNF can facilitate motor axon regeneration into the reimplanted root.44 VEGF is a potent angiogenic peptide that promotes endothelial cell division and enhances neovascularization. In addition, the neurotrophic and neuroprotective effects of VEGF directly influence the behavior of nerve cells.23,45 These findings suggest that etifoxine affects many trophic factors and pathways, thereby offering a new direction for designing therapeutic strategies that promote nerve regeneration. In conclusion, etifoxine greatly enhances the repair of acellular nerve grafts by selectively modulating inflammatory responses to injury as well as increasing expression of neurotrophic factors. Etifoxine fulfills the criteria of a drug that is clinically useful for the treatment of altered peripheral axons.8 These include: (i) easy diffusion into nerve tissues; (ii) 2-fold acceleration of axonal regeneration; (iii) selective modulation of inflammatory responses to injury; (iv) ability to increase expression of neurotrophic factors; (v) suitability for long-term use46,47; and (v) convenience of administration. Given its many benefits, the strategy of combining acellular peripheral nerve graft
FIGURE 8. Effects of etifoxine on mRNA expression. mRNA levels in nerve were normalized to GAPDH mRNA levels. Data are expressed as the mean 6 SEM. *P < 0.05 and **P < 0.01. MUSCLE & NERVE
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implantation with etifoxine treatment may be a good choice for treatment of nerve injuries in humans. The authors are grateful to Weihong Yang for technical assistance. REFERENCES 1. Kline DG, Hudson AR. Vertebral artery compression. J Neurosurg 1995;83:759. 2. Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair and regeneration. Annu Rev Biomed Eng 2003;5:293–347. 3. Wang D, Liu XL, Zhu JK, Jiang L, Hu J, Zhang Y, et al. Bridging small-gap peripheral nerve defects using acellular nerve allograft implanted with autologous bone marrow stromal cells in primates. Brain Res 2008;1188:44–53. 4. Wang D, Liu X L, Zhu J K, Hu J, Jiang L, Zhang Y, et al. Repairing large radial nerve defects by acellular nerve allografts seeded with autologous bone marrow stromal cells in a monkey model. J Neurotrauma 2010;27:1935–1943. 5. Hou SY, Zhang HY, Quan DP, Liu XL, Zhu JK. Tissue-engineered peripheral nerve grafting by differentiated bone marrow stromal cells. Neuroscience 2006;140:101–110. 6. Walsh S, Biernaskie J, Kemp SW, Midha R. Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration. Neuroscience 2009;164:1097–1107. 7. Yu H, Peng J, Guo Q, Zhang L, Li Z, Zhao B, et al. Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor. Microsurgery 2009;29:330–336. 8. Girard C, Liu S, Cadepond F, Adams D, Lacroix C, Verleye M, et al. Etifoxine improves peripheral nerve regeneration and functional recovery. Proc Natl Acad Sci USA 2008;105: 20505–20510. 9. Girard C, Liu S, Adams D, Lacroix C, Sineus M, Boucher C, et al. Axonal regeneration and neuroinflammation: roles for the translocator protein 18 kDa. J Neuroendocrinol 2011;24:71–81. 10. Verleye M, Akwa Y, Liere P, Ladurelle N, Pianos A, Eychenne B, et al. The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain. Pharmacol Biochem Behav 2005;82:712–720. 11. Schlichter R, Rybalchenko V, Poisbeau P, Verleye M, Gillardin J. Modulation of GABAergic synaptic transmission by the nonbenzodiazepine anxiolytic etifoxine. Neuropharmacology 2000;39: 1523–1535. 12. Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere JJ, Lindemann P, et al. Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 2006;27:402–409. 13. Campioli E, Carnevale G, Avallone R, Guerra D, Baraldi M. Morphological and receptorial changes in the epididymal adipose tissue of rats subjected to a stressful stimulus. Obesity (Silver Spring) 2011;19: 703–708. 14. Rechichi M, Salvetti A, Chelli B, Costa B, Da Pozzo E, Spinetti F, et al. TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells. Biochim Biophys Acta 2008;1782:118–125. 15. Lacor P, Benavides J, Ferzaz B. Enhanced expression of the peripheral benzodiazepine receptor (PBR) and its endogenous ligand octadecaneuropeptide (ODN) in the regenerating adult rat sciatic nerve. Neurosci Lett 1996;220:61–65. 16. Karchewski LA, Bloechlinger S, Woolf CJ. Axonal injury-dependent induction of the peripheral benzodiazepine receptor in smalldiameter adult rat primary sensory neurons. Eur J Neurosci 2004;20: 671–683. 17. Torres SR, Frode T, Nardi GM, Vita N, Reeb R, Ferrara P, et al. Antiinflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation. Eur J Pharmacol 2000;408: 199–211. 18. Veiga S, Azcoitia I, Garcia-Segura LM. Ro5–4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity. J Neurosci Res 2005;80:129–137. 19. Ferzaz B, Brault E, Bourliaud G, Robert JP, Poughon G, Claustre Y, et al. SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro4H-pyridazino[4,5-b]indole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair. J Pharmacol Exp Ther 2002;301:1067–1078. 20. Leonelli E, Yague JG, Ballabio M, Azcoitia I, Magnaghi V, Schumacher M, et al. Ro5–4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats. Mech Ageing Dev 2005;126:1159–1163. 21. Mills CD, Bitler JL, Woolf CJ. Role of the peripheral benzodiazepine receptor in sensory neuron regeneration. Mol Cell Neurosci 2005;30: 228–237.
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22. Mills C, Makwana M, Wallace A, Benn S, Schmidt H, Tegeder I, et al. Ro5–4864 promotes neonatal motor neuron survival and nerve regeneration in adult rats. Eur J Neurosci 2008;27:937–946. 23. Sondell M, Lundborg G, Kanje M. Regeneration of the rat sciatic nerve into allografts made acellular through chemical extraction. Brain Res 1998;795:44–54. 24. de Medinaceli L, Freed WJ, Wyatt RJ. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp Neurol 1982;77:634–643. 25. Omura K, Ohbayashi M, Sano M, Omura T, Hasegawa T, Nagano A. The recovery of blood-nerve barrier in crush nerve injury—a quantitative analysis utilizing immunohistochemistry. Brain Res 2004;1001: 13–21. 26. Wang X, Hu W, Cao Y, Yao J, Wu J, Gu X. Dog sciatic nerve regeneration across a 30-mm defect bridged by a chitosan/PGA artificial nerve graft. Brain 2005;128:1897–1910. 27. Liu XL, Arai T, Sondell M, Lundborg G, Kanje M, Dahlin LB. Use of chemically extracted muscle grafts to repair extended nerve defects in rats. Scand J Plast Reconstr Surg Hand Surg 2001;35:337–345. 28. Zeng C, Sheng P, Xie G, Zhu J, Dong P, Quan D. Fabrication of PLLA nanofibrous multi-channel conduits for neural tissue engineering. J Control Release 2011;152(suppl 1):e234–236. 29. Panaite PA, Barakat-Walter I. Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury. J Neurosci Res 2010;88:1751–1763. 30. Moore AM, MacEwan M, Santosa KB, Chenard KE, Ray WZ, Hunter DA, et al. Acellular nerve allografts in peripheral nerve regeneration: a comparative study. Muscle Nerve 2011;44:221–234. 31. Varejao AS, Meek MF, Ferreira AJ, Patricio JA, Cabrita AM. Functional evaluation of peripheral nerve regeneration in the rat: walking track analysis. J Neurosci Methods 2001;108:1–9. 32. Werdin F, Grussinger H, Jaminet P, Kraus A, Manoli T, Danker T, et al. An improved electrophysiological method to study peripheral nerve regeneration in rats. J Neurosci Methods 2009;182:71–77. 33. Radtke C, Aizer AA, Agulian SK, Lankford KL, Vogt PM, Kocsis JD. Transplantation of olfactory ensheathing cells enhances peripheral nerve regeneration after microsurgical nerve repair. Brain Res 2009; 1254:10–17. 34. Delgado-Garcia JM, Del Pozo F, Spencer RF, Baker R. Behavior of neurons in the abducens nucleus of the alert cat—III. Axotomized motoneurons. Neuroscience 1988;24:143–160. 35. Huang J, Hu X, Lu L, Ye Z, Wang Y, Luo Z. Electrical stimulation accelerates motor functional recovery in autograft-repaired 10 mm femoral nerve gap in rats. J Neurotrauma 2009;26:1805–1813. 36. Rosen JM, Pham HN, Hentz VR. Fascicular tubulization: a comparison of experimental nerve repair techniques in the cat. Ann Plast Surg 1989;22:467–478. 37. Aminoff I. [Caution: hemorrhoidal prolapse]. Harefuah 1999;136: 993–996. 38. Snider WD, Johnson EM Jr. Neurotrophic molecules. Ann Neurol 1989;26:489–506. 39. Rich KM, Yip HK, Osborne PA, Schmidt RE, Johnson EM Jr. Role of nerve growth factor in the adult dorsal root ganglia neuron and its response to injury. J Comp Neurol 1984;230:110–118. 40. Taniuchi M, Clark HB, Johnson EM Jr. Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci USA 1986;83:4094–4098. 41. Rich KM, Alexander TD, Pryor JC, Hollowell JP. Nerve growth factor enhances regeneration through silicone chambers. Exp Neurol 1989; 105:162–170. 42. Fine EG, Decosterd I, Papaloizos M, Zurn AD, Aebischer P. GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap. Eur J Neurosci 2002;15:589– 601. 43. Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 1993;260:1130–1132. 44. Bergerot A, Shortland PJ, Anand P, Hunt SP, Carlstedt T. Co-treatment with riluzole and GDNF is necessary for functional recovery after ventral root avulsion injury. Exp Neurol 2004;187:359–366. 45. Storkebaum E, Lambrechts D, Dewerchin M, Moreno-Murciano MP, Appelmans S, Oh H, et al. Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat Neurosci 2005;8:85–92. 46. Micallef J, Soubrouillard C, Guet F, Le Guern ME, Alquier C, Bruguerolle B, et al. A double blind parallel group placebo controlled comparison of sedative and mnesic effects of etifoxine and lorazepam in healthy subjects [corrected]. Fundam Clin Pharmacol 2001;15:209–216. 47. Nguyen N, Fakra E, Pradel V, Jouve E, Alquier C, Le Guern ME, et al. Efficacy of etifoxine compared to lorazepam monotherapy in the treatment of patients with adjustment disorders with anxiety: a double-blind controlled study in general practice. Hum Psychopharmacol 2006;21:139–149.
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Department of Microsurgery and Orthopedic Trauma, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China 2 Department of Physiology, Medical College of Shangtou University, Shantou, China 3 Department of Reproductive Medicine Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Accepted 19 November 2013 ABSTRACT: 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 SpragueDawley 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
Traumatic injury to peripheral nerves may result in considerable loss of sensory and motor function and thereby in decreased quality of life. Several research groups have tried to accelerate regeneration of traumatized nerves through application of microsurgical techniques. However, despite advancements in these techniques, complete recovery is achieved rarely.1,2 Acellular nerve allografts (ANAs) derived from native peripheral nerve retain Abbreviations: ANA, acellular nerve allograft; CMAP, compound muscle action potential; CNS, central nervous system; DRG, dorsal root ganglia; ECM, extracellular matrix; GF, growth factor; GDNF, glia-derived growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H&E, hematoxylin and eosin; NCV, nerve conduction velocity; NGF, nerve growth factor; NF-200, neurofilament-200; PCR, polymerase chain reaction; PB, phosphate buffer; SFI, sciatic functional index; TSPO, translocator protein (18 kDa); VEGF, vascular endothelial growth factor Key words: acellular nerve grafts; etifoxine; nerve regeneration; neurotrophins expression; TSPO This study was supported by grants from National High Technology Research and Development Program of China (2012AA020507), the National Nature Science Grant of China (30700847), 985 program of Sun Yat-sen University (Grant No. 90035-3283312); Specialized Research Fund for the Doctral Program of Higher Education (SRFDP) (Grant No. 20120171120075); and the Doctoral Start-up Project of the Guangdong Nature Science Foundation (S201204006336). The first 2 authors (X.Z. and B.H.) contributed equally to this article. Correspondence to: X. Liu; e-mail: [email protected] C 2013 Wiley Periodicals, Inc. V
Published online 24 November 2013 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24131
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the structure and extracellular matrix (ECM) components of the original nerve and stimulate only a low host immune response. In previous studies, our group demonstrated the usefulness of acellular nerve grafts for repair of small and large defects in many animal models of peripheral nerve injury.3–5 Based on those findings, acellular nerve grafts have been attempted in humans, and a multicenter trial has shown good results (unpublished results). However, some problems remain. The efficiency of nerve cell regeneration in ANAs is low,3,6,7 so we tried to combine grafting with ways to enhance nerve cell regeneration in the graft. Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3, 1-benzoxazine hydrochloride; Stresam, iocodex) is currently under investigation as a therapeutic way to promote neuroprotection, accelerate axonal regeneration, and modulate inflammation.8,9 It is used traditionally as an anxiolytic and anticonvulsant drug. Recent research has shown that etifoxine targets GABA-A receptors and the 18kDa translocator protein TSPO.10,11 TSPO is multifunctional and is localized mainly in the outer mitochondrial membrane.12 In rats, ligand binding stimulates the cholesterol transfer function of TSPO. Campioli et al.13 found that expression of TSPO 18 kDa increases after a stressful stimulus. Overexpression of TSPO increases the motility, transmigration, and proliferation properties of C6 rat glioma cells.14 After peripheral nerve injury, TSPO expression is increased transiently in many cells such as dorsal root ganglia (DRG) neurons, Schwann cells, and macrophages.15,16 Also, TSPO ligands have been shown to exert neuroprotective effects and reduce neural inflammation in the central nervous system (CNS),17,18 protect motoneurons from degeneration after facial nerve injury, reduce sensorimotor deficits after acrylamide intoxication, prevent aging-associated peripheral nerve degeneration,19,20 and stimulate outgrowth of DRG neurites.21 Mills et al. found that the TSPO ligand Ro5–4864 significantly enhances regeneration and functional recovery in the peripheral nervous system of rats.22 Girard et al. found that etifoxine can enhance the repair of peripheral nerve damage MUSCLE & NERVE
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(i.e., accelerate axonal regrowth and improve macrophage responses) in rats.8 All of these experimental observations strongly suggest that TSPO ligands may be used to stimulate axonal regeneration and modulate macrophage activation in damaged peripheral nerves. However, the role of etifoxine in peripheral nerve regeneration and functional recovery after acellular nerve allograft implantation is unknown. Also unknown is whether etifoxine exerts its effect through pathways other than the immune response pathway. We now report that adding etifoxine to the acellular nerve allograft procedure for treatment of peripheral nerve injury improves axonal regrowth and immune responses. Moreover, its administration not only increases the rate but also the extent of functional recovery from sciatic nerve injury. METHODS Surgical Procedures. All animal procedures were approved by the experimental animal administration committee of Sun Yat Sen University. Efforts were made to minimize the suffering of animals during the postsurgery period. Male Sprague-Dawley rats (200–230 g) were anesthetized deeply by intraperitoneal injection of pentobarbital (75 mg/kg). Under aseptic conditions, the skin of the left leg was cut parallel to the femur. The sciatic nerve was exposed by splitting the superficial gluteus muscle. 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. Nerve grafts were sutured microsurgically to the proximal and distal nerve stumps using a 10-0 nylon suture (Sharpoint; Surgical Specialties Corp., Reading, Pennsylvania), with suture of the epineurial sheathes of the sciatic nerve. The rats were divided randomly into 3 groups: (1) an autograft group (n 5 24), in which a 15-mm segment of the sciatic nerve was removed, inverted, and reimplanted into the gap; (2) a simple acellular nerve graft group (n 5 24), in which a 15-mm segment of the sciatic nerve was replaced with an 18-mm acellular nerve graft, with daily intraperitoneal injections of 0.5 ml per 100 g vehicle (1% Tween-80 in 0.9% NaCl solution); and (3) an acellular nerve graft plus etifoxine group (n 5 24), in which the 15-mm segment of the sciatic nerve was replaced with an 18-mm acellular nerve graft, with daily intraperitoneal injections of etifoxine (Batch 285; Biocodex, Gentilly, France) at a dose of 50 mg/kg for 7 days. Etifoxine has been shown to exhibit effects in rats.8 The overlying muscle and skin of the thigh were sutured with 6-0 and 4-0 polyamide sutures. Treatment was started 24 hours after surgery. The animals were allowed to survive up to 12 weeks after surgery. 236
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For nerve graft preparation Wistar rats were killed by intraperitoneal injection with sodium pentobarbital (0.5 ml, 60 mg/ml). The sciatic nerves were excised, cleaned of external debris, and treated with chemical detergents to produce acellular allografts. The method for isolating acellular grafts was based on a procedure developed by Sondell et al.23 Immunohistochemistry. Two weeks after surgery, 6 rats per group were anesthetized with pentobarbital (1.5 g/kg, intraperitoneally) and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at a pH of 7.2–7.4 at 4 C. Rat sciatic nerves were excised, postfixed in 4% paraformaldehyde for 3 hours, dipped in 30% sucrose (in 0.1 M PB) overnight, sectioned (16 lm thickness) in the longitudinal direction on a freezing microtome (Leica CM3050S; Leica, Wetzlar, Germany), and collected into 0.01 M phosphate buffered saline. Immunohistochemical studies were performed using a monoclonal anti–neurofilament 200 (NF200) antibody (Sigma-Aldrich, Tokyo, Japan). The frozen sections were preincubated in 3% hydrogen peroxide and 10% normal rabbit serum for 10 minutes to block nonspecific binding of immunoglobulin, incubated at room temperature overnight with monoclonal NF-200 antibodies (diluted 1:400 in PB), washed 3 times with 0.01 M phosphatebuffered saline (PBS), incubated with Cy3conjugated donkey anti-rabbit IgG (1:300; Jackson ImmunoResearch, West Grove, Pennsylvania), rinsed 3 times with 0.01 M PBS, mounted on gelatin-coated slides, and air-dried. The images of the stained sections were captured with a fluorescence microscope attached to a CCD spot camera (DFC350FX/DMIRB; Leica) and processed with Leica IM50 software. Histological Examination. Four weeks after surgery, 6 rats per group were euthanized for histological examination. The gastrocnemius muscles and repaired nerve segments were harvested in all 3 groups. The muscle samples were postfixed with formalin, embedded in paraffin, and sectioned transversely. The sections were deparaffinized, rehydrated in an alcohol series, and stained with hematoxylin and eosin. For each sample, photographs were taken from 3 random fields and analyzed using ImagePro Plus (version 6.0) software to measure the area of the muscle fibers. Six samples from each group were used for statistical analysis. Toluidine blue staining was performed as described previously.3 Briefly, nerve graft segments were harvested and quickly immersed in 2.5% Nacacodylate–buffered glutaraldehyde solution for 2 MUSCLE & NERVE
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h, fixed for 2 h in 2% Na-cacodylate–buffered osmium tetroxide, serially dehydrated in increasing concentrations of ethanol, infiltrated and embedded in Epon 812 (Ted Pella, Redding, California), sectioned (1 lm thickness), and stained with toluidine blue to evaluate the efficacy of nerve regeneration. Transection was performed in the middle of the nerve graft segments. Analysis of the average number of myelinated axons and fiber diameter was performed using an Olympus BX60 microscope and ImagePro Plus (version 6.0) quantitative morphology software. For each sample, photographs were taken from 3 random fields and analyzed to measure the number and diameter. Six samples from each group were used for statistical analysis. Transmission electron microscopy was employed to evaluate myelin sheath regeneration. Ultrathin sections were stained with lead citrate and uranyl acetate and examined under a Philips CM120 transmission electron microscope equipped with an image acquisition system at 80003 magnification for measuring thickness of myelin sheaths. Photographs from 10 random fields of each ultrathin nerve section were analyzed with ImagePro Plus software. Walking-Track Analysis. Functional nerve regeneration was assessed by calculating the sciatic functional index (SFI).24 Briefly, after the hind feet were dipped in dark ink, rats walked down a wooden corridor. SFI was calculated using the following formula: EPL1NPL ETS1NTS 1 109:53 SFI 5 238:33 NPL NTS EIT 1NIT 28:8 1 13:33 NIT
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 toes; and the intermediary toe spread (IT) was defined as the distance from the second to fourth toes. All 3 measurements were made on the experimental (E) and normal (N) sides. The walking functional test was performed every 2 weeks, beginning at week 2 and ending at week 12. Electrophysiological Testing. Twelve weeks after surgery, 6 rats per group were anesthetized, placed in the prone position for better exposure of the left sciatic nerve, and tested by placement of stimulating crook-shaped silver needle electrodes on the proximal and distal ends of the grafts. Normal compound muscle action potentials (CMAPs) on the contralateral side were also recorded for comEtifoxine in Nerve Regeneration
parison. A personal computer was used to set the conditions, including the frequency and amplitude of the stimulation signal, and recordings were performed using a Nicolet Viking electrodiagnostic system (Nicolet Instrument Corp., Madison, Wisconsin). Digitized data were stored on computer. The peak amplitude and nerve conduction velocity (NCV) were calculated. Real-Time
Quantitative
Polymerase
Chain
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA levels of nerve growth factor (NGF), glia-derived growth factor (GDNF), and vascular endothelial growth factor (VEGF) in the nerve segment. To measure the effect of etifoxine, the etifoxine and saline groups were used in this assay. Primers for rat NGF, GDNF, VEGF, and glyceraldehyde 3phosphate dehydrogenase (GAPDH) were designed using Primer Express v2.0 software (Applied Biosystems, Foster City, California) and obtained at Huirui Bio Technologies (Shanghai, China). One week after surgery, 6 rats per group were euthanized, and their sciatic nerves were stored in RNA-Later (Ambion, Austin, Texas) at 220 C. Total RNA was extracted with Trizol (Invitrogen, Carlsbad, California), purified on RNeasy minicolumns (Qiagen, Valencia, California), and treated with RNase-free DNAse I (Qiagen). The RNA purity (OD260/280 absorption ratio) was determined to be about 1.9–2.0. cDNA was synthesized using a first-strand RT-PCR kit (SuperScript II; Invitrogen). Gene expression was measured by qPCR (MX4000; Stratagene, La Jolla, California) with 50 ng of rat cDNA and 2 3 TaqMan universal PCR master mix (Applied Biosystems) with a 1-step program (95 C for 10 minutes, 95 C for 30 seconds, and 60 C for 1 minute for 50 cycles). Duplicate samples without cDNA (no-template control) for each gene showed no contaminating DNA. Gene expression levels were normalized to GAPDH and were quantified using the comparative critical threshold (Ct) method. Reaction.
Statistical Analysis. All numerical data are given as mean 6 standard error (SE). All results were subjected to statistical analysis using SPSS v11.5 software for Windows (student version). Statistically significant values were defined as P < 0.05. RESULTS Density of Regenerated Axons.
Animals (n 5 6 per group) were euthanized to measure neurofilament protein expression 2 weeks after surgery. Evidence indicates that increased expression of neurofilament proteins reflects early regenerative potential.25 Analysis of immunostained neurofilaments (Fig. 1) demonstrated that expression of MUSCLE & NERVE
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FIGURE 1. Immunohistochemistry analysis of longitudinal sections in the middle of the bridge 2 weeks after surgery. Repair using (A) autologous sciatic nerve, (B) ANA alone, and (C) ANA combined with etifoxine treatment. (D) Neurofilament expression was greater in the autologous group than in the etifoxine group and greater in the etifoxine group than in the ANA group. **P < 0.01 and ***P < 0.001, determined by 2-tailed Student t-tests.
neurofilament proteins was enhanced significantly by etifoxine treatment compared with nontreatment (546,953 6 22,110 vs. 455,760 6 24,979, P < 0.01), but remained significantly lower than expression in the autografts (P < 0.001). Histological Examination. Hematoxylin–eosin (H&E) staining was performed to assess the atrophy of rat gastrocnemius muscle resulting from sciatic nerve damage. The end of the atrophic process is accompanied by gradual recovery of sciatic nerve function.26 The contralateral gastrocnemius with intact nerve (the positive control) had a normal histological appearance with no muscle atrophy (Fig. 2). In contrast, the ipsilateral gastrocnemius muscle was atrophied. The average percentage of muscle fiber area was significantly higher after etifoxine treatment than saline treatment 4 weeks postoperatively (54.1 6 2.3% vs. 66.5 6 1.8%, P < 0.01), confirming a better functional recovery after etifoxine. Examination of the toluidine blue–stained nerve segments at the mid-portion in 3 experimental groups at 8 weeks showed that adding etifoxine greatly enhanced axon regeneration (Fig. 3). Myelinated fibers were of similar size and shape 238
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and were arranged symmetrically in the autograft group, but they were spaced randomly and were less symmetrically shaped in the etifoxine and ANA groups. The number of regenerated axons and axon diameter were larger in the etifoxine group. The number of axons was similar in the etifoxine and autograft groups, suggesting regeneration was achieved in both groups (49,533 6 1245 vs. 54,674 6 2486, P > 0.05). The number of myelinated (toluidine blue positive) axons was significantly higher in the etifoxine group than in the ANA group (40,523 6 1436 vs. 49,533 6 1245, P < 0.001) (Fig. 4A). Although similar in the etifoxine and autograft groups (2.86 6 0.15 vs. 2.69 6 0.21), the average diameter of myelinated fibers was significantly larger in the etifoxine group than in the ANA group (1.93 6 0.33 vs. 2.69 6 0.21, P < 0.05) (Fig. 4B). Transmission electron microscopy revealed regeneration of myelinated fibers in the midportion of the implant 1 month after implantation in each group. The myelinated fibers were more compact and uniform in the etifoxine and autograft groups than in the ANA group. The myelin MUSCLE & NERVE
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FIGURE 2. H&E staining of gastrocnemius muscle. (A) Positive control. (B) Autograft group. (C) ANA group. (D) Etifoxine group.
sheath thickness was greater in the etifoxine group than in the ANA group (1.02 6 0.13 vs. 0.66 6 0.10, P < 0.05), but was similar in the etifoxine and autograft groups (Fig. 5D). Walking-track analysis is a useful technique for evaluating motor functional recovery after sciatic nerve injury. The increase in sciatic nerve functional index (SFI) after treatment indicates peripheral nerve regeneration. As shown in Figure 6, the level of regeneration was significantly higher in the etifoxine group than in the ANA group 8 weeks postoperatively, but still lower than in the autologous group. This result shows that etifoxine treatment enhances functional recovery in ANAs. By 10 weeks postoperatively, functional recovery was almost the same in the etifoxine and autologous groups (Fig. 6).
Functional Results.
Improvement in Electrophysiology. Compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were used to evaluate electrophysiological recovery after surgery. The change in CMAP (% CMAP) was significantly higher in the autograft group (49.6%) than in the ANA (32.3%) and etofoxine (37.8%) groups, but was similar in the ANA and etifoxine groups (Fig. 7A). The Etifoxine in Nerve Regeneration
change in NCV (% NCV) was significantly higher in the autograft group (84.1%) than in the ANA group (56.7%) and significantly higher in the etifoxine group (77.4%) than in the ANA group, but it was similar in the etifoxine and autograft groups (Fig. 7B). These results suggest that etifoxine enhances axonal regeneration. Although etifoxine influences the immune response in rat sciatic nerve, it is unknown whether it influences neurotrophic expression in peripheral nerves. To determine whether etifoxine modulates neurotrophic expression after peripheral nerve injury, the expression of NGF, BDNF, and VEGF was quantified in the regenerating sciatic nerve (Fig. 8). At 7 days after injury, etifoxine treatment had a marked neurotrophic effect (NGF: 1.9-fold increase over control, P < 0.05; GDNF: 2.0-fold increase over control, P < 0.01; VEGF: 1.7-fold increase over control, P < 0.01).
Gene Expression.
DISCUSSION
Girard et al.8 combined etifoxine treatment with silicone tube implantation to repair injured rat sciatic nerve. Silicone or other materials have been used to construct nerve guides for this MUSCLE & NERVE
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FIGURE 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. Thin (1 lm) sections of sciatic nerve specimens were stained with 1% toluidine blue for qualitative examination of the midline of the graft by light microscopy. Groups treated with etifoxine demonstrated more organized neural architecture, closely approximating the autograft, in comparison to the ANA alone group.
purpose.27–29 Nerve graft success depends on various factors: (1) the wall thickness and structural integrity of the graft should be retained to prevent collapse; (2) the materials used to support the graft should be biodegradable and not cytotoxic and provide a suitable matrix for cell adhesion and cell migration; and (3) the grafts should be immunologically tolerated by recipients. ANAs derived from native peripheral nerves retain the
structure and ECM components of the original tissue and stimulate only weak host immune responses. Once implanted, these acellular nerves serve as a natural scaffold into which surrounding cells readily migrate, forming the foundation for new tissue. Thus, acellular nerve implantation is a more promising strategy than other nerve implantation techniques.30 We also believe etifoxine treatment is a good way to improve ANA outcome.
FIGURE 4. (Left) Average diameter of the nerve. (Right) Number of myelinated axons. Error bars indicate mean 6 SEM. Significance of differences was determined by t-test. Results were obtained from 6 rats in each group. *P < 0.05 and **P < 0.001 compared with the etifoxine (ANA1E) and autologous (Auto) groups, determined by 2-tailed Student t-tests. 240
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FIGURE 5. Transmission electron micrographs. (A) Autograft group. (B) ANA group. (C) Etifoxine group. (D) Myelin sheath thickness of each group. Error bars indicate mean 6 SEM. Significance of difference was determined by t-test. *P < 0.05.
Previous reports have shown that muscles may atrophy irreversibly after nerve damage and lose function before axonal regeneration into distal targets. Thus, the speed of regeneration is important in peripheral nerve regeneration. Our results show that in the early stage of nerve regeneration etifoxine treatment accelerated nerve regeneration in ANA. Regeneration of peripheral axons to reach the muscle end-plates weeks or months before atrophy can be very beneficial. In our study, atrophy of gastrocnemius muscles was less pronounced when etifoxine treatment was provided after acellular graft implantation. Histological assessment showed that nerve fibers grew better in the etifoxine group, and the structure of regenerated nerves was similar to that in the autograft group (Fig. 3). In contrast, myelinated nerve fibers in the ANA group were less dense, of uneven size, and less myelinated. H&E stains revealed that gastrocnemius muscle atrophy 3 months after implantation was most obvious in the ANA group with the least percentage of muscle fiber area, suggesting little recovery of nerve function. However, the recovery level in rats treated with etifoxine approached that of the autologous group. In the third part of our study, walking-track analysis was conducted to evaluate function. When measuring experimental nerve recovery, selection of the appropriate methods of evaluation is crucial. In 1982, de Medinaceli et al. first reported that the SFI could be used to evaluate total lower limb Etifoxine in Nerve Regeneration
function, including nerve, muscle, and joint function in rats.24 Walking-track analysis is a noninvasive method to assess sciatic nerve function based on measurement of footprints.23 Because proper walking demands coordinated functions, such as sensory input, motor control, and cortical integration, SFI may be a better measure of function than electrophysiological and histomorphometric measures of axon growth, especially when the research focus is on functional outcome.31 In our study, walking-track analysis findings paralleled histologic findings. Early recovery of SFI is hard to achieve, although histological analyses show good results in
FIGURE 6. Etifoxine treatment improved the functional recovery of transected sciatic nerve. Locomotion was assessed by the walking-track test, and footprints were monitored at 2, 4, 6, 8, 10, and 12 weeks after transection. Improved recovery of locomotion in etifoxine-treated animals was followed by increase in sciatic functional index (SFI), calculated using the de Medinaceli method.23 *P < 0.05 and **P < 0.01, comparing ANA1E with ANA; and ###P < 0.001, comparing ANA1E with Auto, determined by 2-tailed Student t-tests. MUSCLE & NERVE
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FIGURE 7. Electrophysiological evaluation. Nerve conduction velocity (NCV) and compound muscle action potentials (CMAPs) were measured at 12 weeks after sciatic nerve transection. The ratio of nerve conduction latency (B) and CMAP (A) on the injured side compared with the normal side was calculated 12 weeks after injury. *P < 0.05 (n 5 6).
2 weeks, because the recovery of function needs more than just histological recovery. However, improvement was significantly better in the etifoxine and autologous groups than in the ANA group after 8 weeks, but it was not significantly different between the etifoxine and autologous groups, again showing the effectiveness of etifoxine treatment. Electrophysiological analysis is a useful test of functional recovery after nerve injury.32,33 CMAP peak amplitude and NCV decrease after nerve injury.34,35 Electrophysiological recordings demonstrated almost the same level of improvement in nerve conduction velocity in the etifoxine and autologous groups. However, CMAP amplitude was similar in the etifoxine and ANA groups (Fig. 7). Studies suggested that the CMAP amplitude reflects the number of axons reinnervating the muscle and is related to the amount of acetylcholine release, and nerve conduction latency is related inversely to motor function improvement.36,37 Thus, the effect of etifoxine on motor function improvement may be more profound than its effect on acetylcholine release. The relatively small sample size may also account for this result. Etifoxine may also enhance synthesis of neurotrophic factors. Neurotrophic factors are a category of polypeptides or proteins that are released by neurons or non-neuronal cells and have an extensive impact on both the central and peripheral nervous systems.38 In our study, the amount of NGF mRNA was increased after etifoxine treatment. Exogenously applied NGF has been reported to prevent the death of axotomized sensory neurons.39,40 However, the stability of NGF is limited under physiological conditions41,42 and may be reduced further by exposure to organic solvents, shearing, and acidic degradation. This may explain why extrinsic NGF is effective. In our study, NGF was increased endogenously to better promote regeneration. In addition to NGF mRNA, GDNF and VEGF mRNA were also found to increase after etifoxine treatment. GDNF purified from the glial cell line B49 was shown to protect dopaminergic neurons from the rat embryonic midbrain.43 When GDNF 242
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was delivered intrathecally after avulsion and reimplantation of lumbar spinal roots, more motoneurons not only survived but were labeled retrogradely, indicating that GDNF can facilitate motor axon regeneration into the reimplanted root.44 VEGF is a potent angiogenic peptide that promotes endothelial cell division and enhances neovascularization. In addition, the neurotrophic and neuroprotective effects of VEGF directly influence the behavior of nerve cells.23,45 These findings suggest that etifoxine affects many trophic factors and pathways, thereby offering a new direction for designing therapeutic strategies that promote nerve regeneration. In conclusion, etifoxine greatly enhances the repair of acellular nerve grafts by selectively modulating inflammatory responses to injury as well as increasing expression of neurotrophic factors. Etifoxine fulfills the criteria of a drug that is clinically useful for the treatment of altered peripheral axons.8 These include: (i) easy diffusion into nerve tissues; (ii) 2-fold acceleration of axonal regeneration; (iii) selective modulation of inflammatory responses to injury; (iv) ability to increase expression of neurotrophic factors; (v) suitability for long-term use46,47; and (v) convenience of administration. Given its many benefits, the strategy of combining acellular peripheral nerve graft
FIGURE 8. Effects of etifoxine on mRNA expression. mRNA levels in nerve were normalized to GAPDH mRNA levels. Data are expressed as the mean 6 SEM. *P < 0.05 and **P < 0.01. MUSCLE & NERVE
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implantation with etifoxine treatment may be a good choice for treatment of nerve injuries in humans. The authors are grateful to Weihong Yang for technical assistance. REFERENCES 1. Kline DG, Hudson AR. Vertebral artery compression. J Neurosurg 1995;83:759. 2. Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair and regeneration. Annu Rev Biomed Eng 2003;5:293–347. 3. Wang D, Liu XL, Zhu JK, Jiang L, Hu J, Zhang Y, et al. Bridging small-gap peripheral nerve defects using acellular nerve allograft implanted with autologous bone marrow stromal cells in primates. Brain Res 2008;1188:44–53. 4. Wang D, Liu X L, Zhu J K, Hu J, Jiang L, Zhang Y, et al. Repairing large radial nerve defects by acellular nerve allografts seeded with autologous bone marrow stromal cells in a monkey model. J Neurotrauma 2010;27:1935–1943. 5. Hou SY, Zhang HY, Quan DP, Liu XL, Zhu JK. Tissue-engineered peripheral nerve grafting by differentiated bone marrow stromal cells. Neuroscience 2006;140:101–110. 6. Walsh S, Biernaskie J, Kemp SW, Midha R. Supplementation of acellular nerve grafts with skin derived precursor cells promotes peripheral nerve regeneration. Neuroscience 2009;164:1097–1107. 7. Yu H, Peng J, Guo Q, Zhang L, Li Z, Zhao B, et al. Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor. Microsurgery 2009;29:330–336. 8. Girard C, Liu S, Cadepond F, Adams D, Lacroix C, Verleye M, et al. Etifoxine improves peripheral nerve regeneration and functional recovery. Proc Natl Acad Sci USA 2008;105: 20505–20510. 9. Girard C, Liu S, Adams D, Lacroix C, Sineus M, Boucher C, et al. Axonal regeneration and neuroinflammation: roles for the translocator protein 18 kDa. J Neuroendocrinol 2011;24:71–81. 10. Verleye M, Akwa Y, Liere P, Ladurelle N, Pianos A, Eychenne B, et al. The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain. Pharmacol Biochem Behav 2005;82:712–720. 11. Schlichter R, Rybalchenko V, Poisbeau P, Verleye M, Gillardin J. Modulation of GABAergic synaptic transmission by the nonbenzodiazepine anxiolytic etifoxine. Neuropharmacology 2000;39: 1523–1535. 12. Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere JJ, Lindemann P, et al. Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function. Trends Pharmacol Sci 2006;27:402–409. 13. Campioli E, Carnevale G, Avallone R, Guerra D, Baraldi M. Morphological and receptorial changes in the epididymal adipose tissue of rats subjected to a stressful stimulus. Obesity (Silver Spring) 2011;19: 703–708. 14. Rechichi M, Salvetti A, Chelli B, Costa B, Da Pozzo E, Spinetti F, et al. TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells. Biochim Biophys Acta 2008;1782:118–125. 15. Lacor P, Benavides J, Ferzaz B. Enhanced expression of the peripheral benzodiazepine receptor (PBR) and its endogenous ligand octadecaneuropeptide (ODN) in the regenerating adult rat sciatic nerve. Neurosci Lett 1996;220:61–65. 16. Karchewski LA, Bloechlinger S, Woolf CJ. Axonal injury-dependent induction of the peripheral benzodiazepine receptor in smalldiameter adult rat primary sensory neurons. Eur J Neurosci 2004;20: 671–683. 17. Torres SR, Frode T, Nardi GM, Vita N, Reeb R, Ferrara P, et al. Antiinflammatory effects of peripheral benzodiazepine receptor ligands in two mouse models of inflammation. Eur J Pharmacol 2000;408: 199–211. 18. Veiga S, Azcoitia I, Garcia-Segura LM. Ro5–4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity. J Neurosci Res 2005;80:129–137. 19. Ferzaz B, Brault E, Bourliaud G, Robert JP, Poughon G, Claustre Y, et al. SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro4H-pyridazino[4,5-b]indole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair. J Pharmacol Exp Ther 2002;301:1067–1078. 20. Leonelli E, Yague JG, Ballabio M, Azcoitia I, Magnaghi V, Schumacher M, et al. Ro5–4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats. Mech Ageing Dev 2005;126:1159–1163. 21. Mills CD, Bitler JL, Woolf CJ. Role of the peripheral benzodiazepine receptor in sensory neuron regeneration. Mol Cell Neurosci 2005;30: 228–237.
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