http://informahealthcare.com/grf ISSN: 0897-7194 (print), 1029-2292 (electronic) Growth Factors, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/08977194.2014.953630
RESEARCH PAPER
Neural mobilization promotes nerve regeneration by nerve growth factor and myelin protein zero increased after sciatic nerve injury Joyce Teixeira da Silva1, Fabio Martinez dos Santos1,2, Aline Caroline Giardini1, Daniel de Oliveira Martins1, Mara Evany de Oliveira1, Adriano Polican Ciena1, Vanessa Pacciari Gutierrez3, Ii-sei Watanabe1, Luiz Roberto G. de Britto4, and Marucia Chacur1
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Department of Anatomy, Laboratory of Functional Neuroanatomy of Pain, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Department of Health Sciences, University Nove de Julho, Sa˜o Paulo, Brazil, 3Special Laboratory of Pain and Signaling, Butantan Institute, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, and 4Department of Physiology and Biophysics, Laboratory of Cellular Neurobiology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract
Keywords
Neurotrophins are crucial in relation to axonal regrowth and remyelination following injury; and neural mobilization (NM) is a noninvasive therapy that clinically is effective in neuropathic pain treatment, but its mechanisms remains unclear. We examined the effects of NM on the regeneration of sciatic nerve after chronic constriction injury (CCI) in rats. The CCI was performed on adult male rats, submitted to 10 sessions of NM, starting 14 days after CCI. Then, the nerves were analyzed using transmission electron microscopy and western blot for neural growth factor (NGF) and myelin protein zero (MPZ). We observed an increase of NGF and MPZ after CCI and NM. Electron microscopy revealed that CCI-NM samples had high numbers of axons possessing myelin sheaths of normal thickness and less inter-axonal fibrosis than the CCI. These data suggest that NM is effective in facilitating nerve regeneration and NGF and MPZ are involved in this effect.
MPZ, NGF, nerve regeneration, neural mobilization, sciatic nerve
Introduction The neuropathic pain is frequently associated with Wallerian degeneration an auto-destructive process, a reaction of the peripheral nervous system to different kinds of nerve injury (Sommer et al., 1998; Uceyler & Sommer, 2006). Numerous studies have demonstrated that myelin protein zero (MPZ) is important for myelin formation and may also play a role in adult axon regeneration (Schweitzer et al., 2003). The MPZ is an integral membrane glycoprotein synthesized by Schwann cells, represents the major glycoprotein of peripheral nerve myelin and plays a fundamental role in adhesion and compaction of peripheral myelin (D’Urso et al., 1990, 1999; Spiryda, 1998). There is evidence that Schwann cells interact via the p75 neural growth factor (NGF) receptor, inducing regeneration after nerve axotomy (Radtke & Vogt, 2009; Raivich et al., 1991; Taniuchi et al., 1988). NGF belongs to a still growing family of molecules collectively called neurotrophins and that play an important role on survival, growth and neural differentiation (Hanani, 2005). NGF is produced and released
Correspondence: Marucia Chacur, PhD, Department of Anatomy, Laboratory of Functional Neuroanatomy of Pain, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. LineuPrestes, 2415, 05508-900, Sa˜o Paulo, Brazil. Tel: (55) (11) 3091-8452. Fax: (55) (11) 3091-8449. E-mail: [email protected]
History Received 23 June 2014 Revised 6 August 2014 Accepted 6 August 2014 Published online 9 December 2014
by target tissues, and there is a reuptake in a retrograde way to maintain neuronal survival. Topically applied NGF stimulates nerve regeneration and promotes functional recovery in crushed rat sciatic nerves (Chen & Wang, 1995). Peripheral nervous system injuries often lead to physical disabilities that can profoundly alter the patients’ quality of life and may impose enormous psychosocial and economic burdens. The peripheral nerves can be recovered from injury through regeneration of axons, leading to the reinnervation of end organs. Despite of modern surgical techniques, the outcome after peripheral nerve injury remains relatively poor. In order to recover the injured peripheral nerve to the pre-injury level, investigators have tried several approaches, such as, the administration of an electric field (electric stimulation) (Leung et al., 2014), application of stem cells (Franchi et al., 2012) and neurotrophic factors (Wild et al., 2007) to optimize the regenerative process. The neural mobilization (NM) technique is a manual therapy method used by physiotherapists to treat patients with pain of neural origin, such as the compression of the sciatic nerve. NM is a noninvasive technique that has been effective in improving the quality of life of patients with neuropathic pain and diverse pain syndromes (Anandkumar et al., 2012; Dwornik et al., 2007; Ellis & Hing, 2008; Oskay et al., 2010; Veras et al., 2012; Villafane et al., 2013). During the execution of body movements, the conjunctive tissue protects
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the axons from tension and compression forces interfering with peripheral nerves (Zamberlan & Kerppers, 2007). It should be stressed that the propagation of electrical impulses depends also on the elasticity of normal nervous tissue, as a normal component of its neurodynamics (Smaniotto & Fonteque, 2004). The NM technique aims to restore mobility and elasticity of the peripheral nervous system and thus to improve the conditions of patients with various neural injuries (Butler, 1991; Butler & Gifford, 1989; Oskay et al., 2010; Santos, 2004). There are several studies indicating that NM is clinically effective in patients with neuropathies (Scrimshaw & Maher, 2001; Walsh, 2005). However, the literature is scarce when surveyed on the possible processes involved. There are only a few articles showing that joint mobilization is able to decrease pain sensitivity after neural injury or after capsaicin injection to the ankle joint (Bertolini et al., 2011; Sluka & Wright, 2001; Sluka et al., 2006). We recently showed that the NM treatment reverses pain symptoms in chronic constriction injured (CCI) rats and suggest the involvement of glial cells and NGF in such an effect (Martins et al., 2012). The aim of this study was to analyze if NM can change the expression of MPZ, NGF and its influence on nerve regeneration. The latter issue was evaluated by the analysis of the nerve fibers in the sciatic nerve of adult neuropathic rats after CCI injury.
Methods Animals Male Wistar rats, weighing between 180 and 220 g (ca. two months old) were used in all experiments. They were singly housed and maintained on a 12:12 h light/dark cycle. The rats were adapted to the experimental environment three days before the experiments started. All animals were tested during the light cycle at the same time of the day (9:00 am– 12:00 am). All procedures were approved by the Institutional Animal Care Committee of the University of Sa˜o Paulo (protocol number 26 – book number 02/2010). Efforts were made to minimize the number of animals used and their suffering. All animals were submitted to behavioral tests to evaluate sensibility in previous study (Martins et al., 2012). Surgical procedure Chronic constriction injury For the induction of neuropathic pain, chronic constriction of the sciatic nerve was performed as previously described by Bennett & Xie in 1988. In short, rats were anesthetized with halothane (Cristalia, Minas Gerais, Brazil) (Bennett & Xie, 1988). The common sciatic nerve was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic trifurcation (about 7 mm), the nerve was freed of adhering tissue and four ligatures (4.0 chromic gut) were tied loosely around it with about 1-mm spacing. Great care was taken to tie the ligatures, such that the diameter of the nerve was seen to be just barely constricted. The incision was closed in layers. In sham-operated rats, the sciatic nerve was exposed but left unaffected and served as a
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control. Each rat was closely observed during the recovery from anesthesia and then returned to the home cage and was observed carefully during the following 24 h. During the 5-dperiod after CCI, the walking and cage exploration, degree of limping and conditions of the hindpaw, including signs of excessive grooming or autotomy, were all closely observed. NM technique The NM technique used in this study has been described by Butler (1989) and adapted by our laboratory (Martins et al., 2012). Briefly, rats were anesthetized with halothane with a continuous flow of medicinal oxygen throughout the procedure (5 ml/L). After anesthesia, the animals were positioned in the left lateral position to mobilize the right side (ipsilateral to CCI). The right knee joint was then positioned in full extension (at 0 degrees) and remained so throughout the session. Moreover, the right hip joint was bent between 70 and 80 degrees with the knee in extension until obtaining a small resistance induced by stretching the muscles from compartimentum posterius femoris (m. bicepsfemoris, m. semimembranosus and m. semitendinosus). After the therapist felt the resistance, the angle of the joint was maintained. At this time, the ankle joint was angled between 30 and 45 degrees, using the same principle as described before. After all joints are positioned with minimal resistance of those muscles, oscillatory movements are initiated. The right ankle joint was manipulated in dorsiflexion (30–45 ) by approximately 20 oscillations per minute during 2 min, followed by a 25-s pause for rest. The treatment took place for 10 minutes, and in the last minute, the cervical spine was fully flexed, with the purpose of tensioning the entire neuraxis (Lew & Briggs, 1997). Treatment with the NM technique started 14 d after injury or sham procedure, and the NM sessions was applied every other day, for a total of 10 sessions. Western blot Western blot analyses were performed on samples from individual animals. Neuropathic (CCI), sham and naive rats were sacrificed by decapitation under light isoflurane anesthesia, and the affected sciatic nerve was quickly removed and homogenized in an extraction buffer containing 100 mM Tris, pH 7.4, 10 mM EDTA, 2 mM PMSF and 10 mg/mL aprotinin. After extraction, the homogenates were centrifuged at 11.5g for 20 min, and the protein concentration of the supernatant was determined using the Bradford protein assay with albumin as a standard (Bio-Rad, Hercules, CA) (Bradford, 1976). Samples containing 75 mg protein were loaded on a 12% acrylamide gel and electrotransferred to nitrocellulose membranes using a Bio-Rad miniature transfer apparatus during 1.5 h at 120 V. After transfer, the membranes were treated for 2 h at room temperature with a blocking solution containing 5% powdered milk, washed and incubated overnight at 4 C with rat monoclonal primary antibody against NGF (F30, 1:1000; Santa Cruz Biotechnology, Dallas, TX) and rabbit polyclonal to MPZ (1:1000; Abcam, Cambridge, MA) (Briani et al., 2008; Jang & Svaren, 2009). The membranes were then washed and incubated for 2 h at room temperature with a peroxidase-conjugated, anti-rat (ZIMED
DOI: 10.3109/08977194.2014.953630
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Laboratories Inc., San Francisco, CA) and anti-rabbit secondary antibodies, diluted 1:5000 (GE Healthcare, Pittsburgh, PA). The immunostained bands were corrected by the optical density of b-actin (1:10,000, Sigma-Aldrich, St. Louis, MO), considering samples from control animals as the standard for normalization of the results. The specifically bound antibody was visualized using a chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). The blot was analyzed densitometrically using NIH-Scion Image 4.0.2 and quantified by optical densitometry of the developed autoradiographs (Scion Corporation, Frederick, MD). Figures were mounted with Adobe Photoshop CS.
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Transmission electron microscopy The animals were intraperitoneally anesthetized with urethane (3 g/Kg) and perfused with a modified Karnovsky fixative solution (containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4) (Ciena et al., 2012). After dissecting the affected sciatic nerve (2 mm distal to the constriction), samples (3 mm3) were post fixed in a 1% osmium tetroxide solution at 4 C and subsequently immersed in a 5% uranyl acetate aqueous solution at room temperature. Next, the samples were dehydrated in an increasing alcohol series, immersed in propylene oxide and embedded in Spur resin. Semi-thin sections were cut with a Reichert Ultra CutÕ ultra-microtome and stained with a 1% toluidine blue solution to test the region to be analyzed. Next, ultrathin 60-nm sections were cut, collected on 200 ‘‘mesh’’ copper grids (SigmaÕ ) and contrasted with a 4% uranyl acetate solution and 0.4% lead citrate solution according to Watanabe & Yamada (1983). The grids were observed with a Jeol 1010 transmission electron microscope (Peabody, MA). Relative quantification of the EM was done by ImageJ program (NIH, Bethesda, MD) (Schneider et al., 2012) in order to measure the morphometric data of interest (averages). The diameter of nerve fiber, axon diameter and myelin sheath thickness was evaluated, respectively, to control, Sham, CCI and CCI + MOB groups. The G-ratios was also calculated by dividing the diameter of the axon by fiber diameter, respectively, to control, Sham, CCI and CCI + MOB groups. Statistical analysis Results are presented as the mean ± SEM. Statistical analyses of data were generated using GraphPAd Prism, version 4.02 (Graph-Pad Software Inc., San Diego, CA). Statistical comparison of more than two groups was performed using analysis of variance, differences between means were tested by Tukey test. In all cases, p50.05 was considered statistically significant (Snedecor et al., 1946).
Results Effects of NM on NGF expression A single NGF-positive band was observed in sciatic nerve extracts from all groups analyzed. Figure 1 shows an increase of NGF protein levels (72%) after CCI in relation to naive rats, taken as controls. After NM treatment (CCI-NM), we observed an increase (124%) of NGF expression when compared to naive animals. Regarding the comparison between CCI and CCI NM groups, we could observe an
Figure 1. Densitometric analysis of NGF levels in the sciatic nerve after CCI injury. The normalized average between sham and experimental groups (CCI) is reported. Data for naive animals were taken as 100%. Data are reported as mean ± SEM of six animals per group. *p50.05 compared to CCI animals.
Figure 2. Densitometric analysis of MPZ levels in the sciatic nerve after CCI injury. The normalized average between sham and experimental groups (CCI) is reported. Data for naive animals were taken as 100%. Data are reported as mean ± SEM of six animals per group. *p50.05 compared to CCI animals.
increase of 48% (p50.01) in the group treated with NM. No statistical differences of NGF expression were observed between sham-NM and naive (p40.05) or between sham and sham-NM animals (p40.05) (data not shown). Effects of NM on MPZ expression Similar results were observed when we analyzed the MPZpositive band in the sciatic nerve. Our results showed an increase of MPZ levels (115%) after CCI injury when compared to naive animals (Figure 2). After NM treatment, we observed an enhancement of MPZ expression (207% above the control). Regarding the comparison between CCI and CCI NM groups, our result was the same as observed for NGF protein expression; we observed an increase of 42% (p50.01) in CCI NM group when compared with CCI group. No difference was observed between naive and sham-NM (p40.05) or between sham and sham-NM (p40.05) (data not shown). No differences were observed for b-actin between control and experimental sides at any of the time points tested (Figures 1 and 2).
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Figure 3. Transmission electron photomicrographs of the sciatic nerve. The naı¨ve (A and B) and sham operated (C and D) groups shown regular distribution of the unmyelinated (arrowheads), myelinated fibers (arrows) and Schwann cell nuclei (*). In the CCI group (E and F), the structure of the nerve was severely damaged characterized by Wallerian degeneration, showing axonal degeneration (arrowheads), few regeneration sprouts (*) and activated macrophages filled with lipid droplets (arrows). In the CCI NM group (G and H) revealed unmyelinated (arrowheads), myelinated (larger arrows) fibers regenerated and in the process regeneration. We also noted sprouts more often clustered in polyaxonal pockets (small arrows). Scale Bars: A, C–E and G: 10 mm; B and F: 5 mm; and H: 2 mm.
Transmission electron microscopy of the sciatic nerve The ultrastructural morphology of sciatic nerves (2 mm distal to the constriction) was observed in naive, sham, CCI and CCI NM groups through transverse sections. The naive and sham groups demonstrated essentially intact fibers, with similar distribution between large and small diameters of unmyelinated and myelinated fibers and the Schwann cell nuclei (Figure 3A–D). In the CCI group, the internal structure of the nerve was severely damaged by Wallerian degeneration, which is characterized by the demyelination and axonal degeneration, conversion of the axonal cytoskeleton to granular debris, undigested myelin debris, few regeneration sprouts and active macrophages filled with lipid droplets (Figure 3E and F). After NM, CCI rats presented evident axonal regeneration, with enhanced amount of regenerated fibers and in process of regeneration, clearance of myelin debris and regenerating sprouts. We also noted polyaxonal pockets aggregating sprouts and a normal distribution of neurofilaments (Figure 3G and H). In order to measure the morphometric data of interest (averages), relative quantification of the EM was done (Table 1).
Discussion We demonstrated an increase of NGF and MPZ expression in animals with CCI and CCI-NM when compared to naive animals. NGF plays a very important role on neuronal survival differentiation and growth (Levi-Montalcini, 1987; Pezet & McMahon, 2006). Several neurotrophic factors as NGF, BDNF and GDNF allow axon regeneration in the sciatic nerve (Fine et al., 2002; Gordon, 2009, 2010; Markus et al., 2001; Terenghi, 1999); however, the NGF in the periphery elicits its effect by upregulating other neurotrophins, such as BDNF, which elicits its effect in the spinal cord by acting on NMDA receptors in cells of the superficial dorsal horn. Therefore, studies of the role of NGF in peripheral nervous system help us for a better understanding of pain modulation
Table 1. The ultrastructural morphology measurement of sciatic nerves.
Group Control Sham CCI CCI + MOB
Nerve fiber diameter
Axon diameter
Myelin sheath thickness
G-ratios
8.5 9.8 2.3 4.4
4.7 5.5 1.8 2.9
1.9 1.8 0.37 0.75
0.5 0.6 0.7 0.6
The nerve fiber diameter, axon diameter, myelin sheath thickness and the G-ratios (calculated by dividing the diameter of the axon by fiber diameter) in all groups were evaluated.
(Boyce & Mendell, 2014). Our data demonstrate an increase of NGF in the sciatic nerve of animals with CCI. After treatment with NM, we observed a greater increase, 52%, of NGF when compared to the CCI group. Our findings corroborate studies showing the participation of NGF in sciatic nerve CCI, indicating its involvement in regeneration (Chen & Wang, 1995; Nishi et al., 2004). Moreover, several studies report that the increased of NFG in dorsal root ganglion (DRG) induced hyperalgesia in rats. First, because NGF enhances the expression of its own receptor, TrkA and also NGF has been shown to enhance the expression of substance P and CGRP in the neurons of C fibers, thus sensitizing neurons to hyperalgesia (Chu et al., 2011; Obata et al., 2002). Beyond that, systemic and peripheral administration of NGF induces nociception (Hanani, 2005; Pannese & Procacci, 2002; Ro et al., 1999; Zhou et al., 1999), and the administration of anti-NGF antibodies prevented the hyperalgesia in rats (Cheng et al., 2009; Wild et al., 2007). A recent study, demonstrated the reversion of hyperalgesia after joint mobilization in a model of sciatic nerve crush injury (Martins et al., 2011). In a previous study, from our group, we demonstrated that NM sessions in CCI rats decreases NGF into DRG and reverses the behavioral effects of neuropathic pain, suggesting the involvement of the DRG-expressed NGF with neuropathic
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DOI: 10.3109/08977194.2014.953630
pain and after NM (for revision see Santos et al., 2012) (Martins et al., 2012). Similar results were observed when we analyzed the MPZpositive band in the sciatic nerve. Our results showed an increase of MPZ levels after CCI injury when compared to naive animals and a much higher increase of MPZ after NM treatment (207% above the control). Many studies have demonstrated that MPZ is important for myelin formation and may also play a role in adult axon regeneration (Schweitzer et al., 2003) and mutations of MPZ appear disrupt myelination (Scherer, 1997; Shy, 2006). To determine whether NM accelerated axon outgrowth or the rate of axon regeneration after nerve injury, we evaluated the effect of NM on axon morphology, as determined by electron microscopy. We found that NM helped to improve the myelination axons. Thus, due to trophic (survival- and growth-promoting) effects of NGF (Binder & Scharfman, 2004) and MPZ increased NM appeared to promote the sciatic regeneration by increase of myelinated axons after injury from CCI. These increases coincide with the tissue regeneration (increase of myelin sheath and fibers in animals treated with NM, by transmission electron microscopy). Ankle joint mobilization in rats with neuropathic pain increases sciatic nerve regeneration and inhibits the hyperalgesia, through the possibly inhibition of glial activation in the dorsal horn of the spinal cord (Martins et al., 2011). Our present data are in agreement with those results.
Conclusions In summary, our data reveal that NM sessions are able to improve axonal regeneration in sciatic nerve and its mechanism could be by NGF and MPZ upregulation after a physiotherapy treatment, showing the relevance of a nonpharmacological intervention.
Declaration of interest No competing financial interests exist. This study was supported by FAPESP (2011/22268-0), CNPq, CAPES (Brazil).
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Sommer C, Schmidt C, George A. Hyperalgesia in experimental neuropathy is dependent on the TNF receptor 1. Exp Neurol 1998; 151:138–142. Spiryda LB. Myelin protein zero and membrane adhesion. J Neurosci Res 1998;54:137–146. Taniuchi M, Clark HB, Schweitzer JB, Johnson EM Jr. Expression of nerve growth factor receptors by Schwann cells of axotomized peripheral nerves: Ultrastructural location, suppression by axonal contact, and binding properties. J Neurosci 1988;8:664–681. Terenghi G. Peripheral nerve regeneration and neurotrophic factors. J Anat 1999;194:1–14. Uceyler N, Sommer C. Wallerian degeneration and neuropathic pain. Drug Discov Today: Dis Mech 2006;3:351–356. Veras LS, Vale RG, Mello DB, Castro JA, Lima V, Trott A, Dantas EH. Electromyography function, disability degree, and pain in leprosy patients undergoing neural mobilization treatment. Rev Soc Bras Med Trop 2012;45:83–88. Villafane JH, Cleland JA, Fernandez-de-Las-Penas C. The effectiveness of a manual therapy and exercise protocol in patients with thumb carpometacarpal osteoarthritis: A randomized controlled trial. J Orthop Sports Phys Ther 2013;43:204–213. Walsh MT. Upper limb neural tension testing and mobilization. Fact, fiction, and a practical approach. J Hand Ther 2005;18:241–258. Watanabe I, Yamada E. The fine structure of lamellated nerve endings found in the rat gingiva. Arch Histol Jpn 1983;46:173–182. Wild KD, Bian D, Zhu D, Davis J, Bannon AW, Zhang TJ, Louis JC. Antibodies to nerve growth factor reverse established tactile allodynia in rodent models of neuropathic pain without tolerance. J Pharmacol Exp Ther 2007;322:282–287. Zamberlan LA, Kerppers II. Mobilizac¸a˜o neural como um recurso fisioterapeˆutico na reabilitac¸a˜o de pacientes com acidente vascular encefa´lico. Salus 2007;2:185–191. Zhou XF, Deng YS, Chie E, Xue Q, Zhong JH, McLachlan EM, Rush RA, Xian CJ. Satellite-cell-derived nerve growth factor and neurotrophin-3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat. Eur J Neurosci 1999;11:1711–1722.
RESEARCH PAPER
Neural mobilization promotes nerve regeneration by nerve growth factor and myelin protein zero increased after sciatic nerve injury Joyce Teixeira da Silva1, Fabio Martinez dos Santos1,2, Aline Caroline Giardini1, Daniel de Oliveira Martins1, Mara Evany de Oliveira1, Adriano Polican Ciena1, Vanessa Pacciari Gutierrez3, Ii-sei Watanabe1, Luiz Roberto G. de Britto4, and Marucia Chacur1
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Department of Anatomy, Laboratory of Functional Neuroanatomy of Pain, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Department of Health Sciences, University Nove de Julho, Sa˜o Paulo, Brazil, 3Special Laboratory of Pain and Signaling, Butantan Institute, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, and 4Department of Physiology and Biophysics, Laboratory of Cellular Neurobiology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Abstract
Keywords
Neurotrophins are crucial in relation to axonal regrowth and remyelination following injury; and neural mobilization (NM) is a noninvasive therapy that clinically is effective in neuropathic pain treatment, but its mechanisms remains unclear. We examined the effects of NM on the regeneration of sciatic nerve after chronic constriction injury (CCI) in rats. The CCI was performed on adult male rats, submitted to 10 sessions of NM, starting 14 days after CCI. Then, the nerves were analyzed using transmission electron microscopy and western blot for neural growth factor (NGF) and myelin protein zero (MPZ). We observed an increase of NGF and MPZ after CCI and NM. Electron microscopy revealed that CCI-NM samples had high numbers of axons possessing myelin sheaths of normal thickness and less inter-axonal fibrosis than the CCI. These data suggest that NM is effective in facilitating nerve regeneration and NGF and MPZ are involved in this effect.
MPZ, NGF, nerve regeneration, neural mobilization, sciatic nerve
Introduction The neuropathic pain is frequently associated with Wallerian degeneration an auto-destructive process, a reaction of the peripheral nervous system to different kinds of nerve injury (Sommer et al., 1998; Uceyler & Sommer, 2006). Numerous studies have demonstrated that myelin protein zero (MPZ) is important for myelin formation and may also play a role in adult axon regeneration (Schweitzer et al., 2003). The MPZ is an integral membrane glycoprotein synthesized by Schwann cells, represents the major glycoprotein of peripheral nerve myelin and plays a fundamental role in adhesion and compaction of peripheral myelin (D’Urso et al., 1990, 1999; Spiryda, 1998). There is evidence that Schwann cells interact via the p75 neural growth factor (NGF) receptor, inducing regeneration after nerve axotomy (Radtke & Vogt, 2009; Raivich et al., 1991; Taniuchi et al., 1988). NGF belongs to a still growing family of molecules collectively called neurotrophins and that play an important role on survival, growth and neural differentiation (Hanani, 2005). NGF is produced and released
Correspondence: Marucia Chacur, PhD, Department of Anatomy, Laboratory of Functional Neuroanatomy of Pain, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. LineuPrestes, 2415, 05508-900, Sa˜o Paulo, Brazil. Tel: (55) (11) 3091-8452. Fax: (55) (11) 3091-8449. E-mail: [email protected]
History Received 23 June 2014 Revised 6 August 2014 Accepted 6 August 2014 Published online 9 December 2014
by target tissues, and there is a reuptake in a retrograde way to maintain neuronal survival. Topically applied NGF stimulates nerve regeneration and promotes functional recovery in crushed rat sciatic nerves (Chen & Wang, 1995). Peripheral nervous system injuries often lead to physical disabilities that can profoundly alter the patients’ quality of life and may impose enormous psychosocial and economic burdens. The peripheral nerves can be recovered from injury through regeneration of axons, leading to the reinnervation of end organs. Despite of modern surgical techniques, the outcome after peripheral nerve injury remains relatively poor. In order to recover the injured peripheral nerve to the pre-injury level, investigators have tried several approaches, such as, the administration of an electric field (electric stimulation) (Leung et al., 2014), application of stem cells (Franchi et al., 2012) and neurotrophic factors (Wild et al., 2007) to optimize the regenerative process. The neural mobilization (NM) technique is a manual therapy method used by physiotherapists to treat patients with pain of neural origin, such as the compression of the sciatic nerve. NM is a noninvasive technique that has been effective in improving the quality of life of patients with neuropathic pain and diverse pain syndromes (Anandkumar et al., 2012; Dwornik et al., 2007; Ellis & Hing, 2008; Oskay et al., 2010; Veras et al., 2012; Villafane et al., 2013). During the execution of body movements, the conjunctive tissue protects
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the axons from tension and compression forces interfering with peripheral nerves (Zamberlan & Kerppers, 2007). It should be stressed that the propagation of electrical impulses depends also on the elasticity of normal nervous tissue, as a normal component of its neurodynamics (Smaniotto & Fonteque, 2004). The NM technique aims to restore mobility and elasticity of the peripheral nervous system and thus to improve the conditions of patients with various neural injuries (Butler, 1991; Butler & Gifford, 1989; Oskay et al., 2010; Santos, 2004). There are several studies indicating that NM is clinically effective in patients with neuropathies (Scrimshaw & Maher, 2001; Walsh, 2005). However, the literature is scarce when surveyed on the possible processes involved. There are only a few articles showing that joint mobilization is able to decrease pain sensitivity after neural injury or after capsaicin injection to the ankle joint (Bertolini et al., 2011; Sluka & Wright, 2001; Sluka et al., 2006). We recently showed that the NM treatment reverses pain symptoms in chronic constriction injured (CCI) rats and suggest the involvement of glial cells and NGF in such an effect (Martins et al., 2012). The aim of this study was to analyze if NM can change the expression of MPZ, NGF and its influence on nerve regeneration. The latter issue was evaluated by the analysis of the nerve fibers in the sciatic nerve of adult neuropathic rats after CCI injury.
Methods Animals Male Wistar rats, weighing between 180 and 220 g (ca. two months old) were used in all experiments. They were singly housed and maintained on a 12:12 h light/dark cycle. The rats were adapted to the experimental environment three days before the experiments started. All animals were tested during the light cycle at the same time of the day (9:00 am– 12:00 am). All procedures were approved by the Institutional Animal Care Committee of the University of Sa˜o Paulo (protocol number 26 – book number 02/2010). Efforts were made to minimize the number of animals used and their suffering. All animals were submitted to behavioral tests to evaluate sensibility in previous study (Martins et al., 2012). Surgical procedure Chronic constriction injury For the induction of neuropathic pain, chronic constriction of the sciatic nerve was performed as previously described by Bennett & Xie in 1988. In short, rats were anesthetized with halothane (Cristalia, Minas Gerais, Brazil) (Bennett & Xie, 1988). The common sciatic nerve was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic trifurcation (about 7 mm), the nerve was freed of adhering tissue and four ligatures (4.0 chromic gut) were tied loosely around it with about 1-mm spacing. Great care was taken to tie the ligatures, such that the diameter of the nerve was seen to be just barely constricted. The incision was closed in layers. In sham-operated rats, the sciatic nerve was exposed but left unaffected and served as a
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control. Each rat was closely observed during the recovery from anesthesia and then returned to the home cage and was observed carefully during the following 24 h. During the 5-dperiod after CCI, the walking and cage exploration, degree of limping and conditions of the hindpaw, including signs of excessive grooming or autotomy, were all closely observed. NM technique The NM technique used in this study has been described by Butler (1989) and adapted by our laboratory (Martins et al., 2012). Briefly, rats were anesthetized with halothane with a continuous flow of medicinal oxygen throughout the procedure (5 ml/L). After anesthesia, the animals were positioned in the left lateral position to mobilize the right side (ipsilateral to CCI). The right knee joint was then positioned in full extension (at 0 degrees) and remained so throughout the session. Moreover, the right hip joint was bent between 70 and 80 degrees with the knee in extension until obtaining a small resistance induced by stretching the muscles from compartimentum posterius femoris (m. bicepsfemoris, m. semimembranosus and m. semitendinosus). After the therapist felt the resistance, the angle of the joint was maintained. At this time, the ankle joint was angled between 30 and 45 degrees, using the same principle as described before. After all joints are positioned with minimal resistance of those muscles, oscillatory movements are initiated. The right ankle joint was manipulated in dorsiflexion (30–45 ) by approximately 20 oscillations per minute during 2 min, followed by a 25-s pause for rest. The treatment took place for 10 minutes, and in the last minute, the cervical spine was fully flexed, with the purpose of tensioning the entire neuraxis (Lew & Briggs, 1997). Treatment with the NM technique started 14 d after injury or sham procedure, and the NM sessions was applied every other day, for a total of 10 sessions. Western blot Western blot analyses were performed on samples from individual animals. Neuropathic (CCI), sham and naive rats were sacrificed by decapitation under light isoflurane anesthesia, and the affected sciatic nerve was quickly removed and homogenized in an extraction buffer containing 100 mM Tris, pH 7.4, 10 mM EDTA, 2 mM PMSF and 10 mg/mL aprotinin. After extraction, the homogenates were centrifuged at 11.5g for 20 min, and the protein concentration of the supernatant was determined using the Bradford protein assay with albumin as a standard (Bio-Rad, Hercules, CA) (Bradford, 1976). Samples containing 75 mg protein were loaded on a 12% acrylamide gel and electrotransferred to nitrocellulose membranes using a Bio-Rad miniature transfer apparatus during 1.5 h at 120 V. After transfer, the membranes were treated for 2 h at room temperature with a blocking solution containing 5% powdered milk, washed and incubated overnight at 4 C with rat monoclonal primary antibody against NGF (F30, 1:1000; Santa Cruz Biotechnology, Dallas, TX) and rabbit polyclonal to MPZ (1:1000; Abcam, Cambridge, MA) (Briani et al., 2008; Jang & Svaren, 2009). The membranes were then washed and incubated for 2 h at room temperature with a peroxidase-conjugated, anti-rat (ZIMED
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Laboratories Inc., San Francisco, CA) and anti-rabbit secondary antibodies, diluted 1:5000 (GE Healthcare, Pittsburgh, PA). The immunostained bands were corrected by the optical density of b-actin (1:10,000, Sigma-Aldrich, St. Louis, MO), considering samples from control animals as the standard for normalization of the results. The specifically bound antibody was visualized using a chemiluminescence kit (Amersham Biosciences, Piscataway, NJ). The blot was analyzed densitometrically using NIH-Scion Image 4.0.2 and quantified by optical densitometry of the developed autoradiographs (Scion Corporation, Frederick, MD). Figures were mounted with Adobe Photoshop CS.
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Transmission electron microscopy The animals were intraperitoneally anesthetized with urethane (3 g/Kg) and perfused with a modified Karnovsky fixative solution (containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4) (Ciena et al., 2012). After dissecting the affected sciatic nerve (2 mm distal to the constriction), samples (3 mm3) were post fixed in a 1% osmium tetroxide solution at 4 C and subsequently immersed in a 5% uranyl acetate aqueous solution at room temperature. Next, the samples were dehydrated in an increasing alcohol series, immersed in propylene oxide and embedded in Spur resin. Semi-thin sections were cut with a Reichert Ultra CutÕ ultra-microtome and stained with a 1% toluidine blue solution to test the region to be analyzed. Next, ultrathin 60-nm sections were cut, collected on 200 ‘‘mesh’’ copper grids (SigmaÕ ) and contrasted with a 4% uranyl acetate solution and 0.4% lead citrate solution according to Watanabe & Yamada (1983). The grids were observed with a Jeol 1010 transmission electron microscope (Peabody, MA). Relative quantification of the EM was done by ImageJ program (NIH, Bethesda, MD) (Schneider et al., 2012) in order to measure the morphometric data of interest (averages). The diameter of nerve fiber, axon diameter and myelin sheath thickness was evaluated, respectively, to control, Sham, CCI and CCI + MOB groups. The G-ratios was also calculated by dividing the diameter of the axon by fiber diameter, respectively, to control, Sham, CCI and CCI + MOB groups. Statistical analysis Results are presented as the mean ± SEM. Statistical analyses of data were generated using GraphPAd Prism, version 4.02 (Graph-Pad Software Inc., San Diego, CA). Statistical comparison of more than two groups was performed using analysis of variance, differences between means were tested by Tukey test. In all cases, p50.05 was considered statistically significant (Snedecor et al., 1946).
Results Effects of NM on NGF expression A single NGF-positive band was observed in sciatic nerve extracts from all groups analyzed. Figure 1 shows an increase of NGF protein levels (72%) after CCI in relation to naive rats, taken as controls. After NM treatment (CCI-NM), we observed an increase (124%) of NGF expression when compared to naive animals. Regarding the comparison between CCI and CCI NM groups, we could observe an
Figure 1. Densitometric analysis of NGF levels in the sciatic nerve after CCI injury. The normalized average between sham and experimental groups (CCI) is reported. Data for naive animals were taken as 100%. Data are reported as mean ± SEM of six animals per group. *p50.05 compared to CCI animals.
Figure 2. Densitometric analysis of MPZ levels in the sciatic nerve after CCI injury. The normalized average between sham and experimental groups (CCI) is reported. Data for naive animals were taken as 100%. Data are reported as mean ± SEM of six animals per group. *p50.05 compared to CCI animals.
increase of 48% (p50.01) in the group treated with NM. No statistical differences of NGF expression were observed between sham-NM and naive (p40.05) or between sham and sham-NM animals (p40.05) (data not shown). Effects of NM on MPZ expression Similar results were observed when we analyzed the MPZpositive band in the sciatic nerve. Our results showed an increase of MPZ levels (115%) after CCI injury when compared to naive animals (Figure 2). After NM treatment, we observed an enhancement of MPZ expression (207% above the control). Regarding the comparison between CCI and CCI NM groups, our result was the same as observed for NGF protein expression; we observed an increase of 42% (p50.01) in CCI NM group when compared with CCI group. No difference was observed between naive and sham-NM (p40.05) or between sham and sham-NM (p40.05) (data not shown). No differences were observed for b-actin between control and experimental sides at any of the time points tested (Figures 1 and 2).
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Figure 3. Transmission electron photomicrographs of the sciatic nerve. The naı¨ve (A and B) and sham operated (C and D) groups shown regular distribution of the unmyelinated (arrowheads), myelinated fibers (arrows) and Schwann cell nuclei (*). In the CCI group (E and F), the structure of the nerve was severely damaged characterized by Wallerian degeneration, showing axonal degeneration (arrowheads), few regeneration sprouts (*) and activated macrophages filled with lipid droplets (arrows). In the CCI NM group (G and H) revealed unmyelinated (arrowheads), myelinated (larger arrows) fibers regenerated and in the process regeneration. We also noted sprouts more often clustered in polyaxonal pockets (small arrows). Scale Bars: A, C–E and G: 10 mm; B and F: 5 mm; and H: 2 mm.
Transmission electron microscopy of the sciatic nerve The ultrastructural morphology of sciatic nerves (2 mm distal to the constriction) was observed in naive, sham, CCI and CCI NM groups through transverse sections. The naive and sham groups demonstrated essentially intact fibers, with similar distribution between large and small diameters of unmyelinated and myelinated fibers and the Schwann cell nuclei (Figure 3A–D). In the CCI group, the internal structure of the nerve was severely damaged by Wallerian degeneration, which is characterized by the demyelination and axonal degeneration, conversion of the axonal cytoskeleton to granular debris, undigested myelin debris, few regeneration sprouts and active macrophages filled with lipid droplets (Figure 3E and F). After NM, CCI rats presented evident axonal regeneration, with enhanced amount of regenerated fibers and in process of regeneration, clearance of myelin debris and regenerating sprouts. We also noted polyaxonal pockets aggregating sprouts and a normal distribution of neurofilaments (Figure 3G and H). In order to measure the morphometric data of interest (averages), relative quantification of the EM was done (Table 1).
Discussion We demonstrated an increase of NGF and MPZ expression in animals with CCI and CCI-NM when compared to naive animals. NGF plays a very important role on neuronal survival differentiation and growth (Levi-Montalcini, 1987; Pezet & McMahon, 2006). Several neurotrophic factors as NGF, BDNF and GDNF allow axon regeneration in the sciatic nerve (Fine et al., 2002; Gordon, 2009, 2010; Markus et al., 2001; Terenghi, 1999); however, the NGF in the periphery elicits its effect by upregulating other neurotrophins, such as BDNF, which elicits its effect in the spinal cord by acting on NMDA receptors in cells of the superficial dorsal horn. Therefore, studies of the role of NGF in peripheral nervous system help us for a better understanding of pain modulation
Table 1. The ultrastructural morphology measurement of sciatic nerves.
Group Control Sham CCI CCI + MOB
Nerve fiber diameter
Axon diameter
Myelin sheath thickness
G-ratios
8.5 9.8 2.3 4.4
4.7 5.5 1.8 2.9
1.9 1.8 0.37 0.75
0.5 0.6 0.7 0.6
The nerve fiber diameter, axon diameter, myelin sheath thickness and the G-ratios (calculated by dividing the diameter of the axon by fiber diameter) in all groups were evaluated.
(Boyce & Mendell, 2014). Our data demonstrate an increase of NGF in the sciatic nerve of animals with CCI. After treatment with NM, we observed a greater increase, 52%, of NGF when compared to the CCI group. Our findings corroborate studies showing the participation of NGF in sciatic nerve CCI, indicating its involvement in regeneration (Chen & Wang, 1995; Nishi et al., 2004). Moreover, several studies report that the increased of NFG in dorsal root ganglion (DRG) induced hyperalgesia in rats. First, because NGF enhances the expression of its own receptor, TrkA and also NGF has been shown to enhance the expression of substance P and CGRP in the neurons of C fibers, thus sensitizing neurons to hyperalgesia (Chu et al., 2011; Obata et al., 2002). Beyond that, systemic and peripheral administration of NGF induces nociception (Hanani, 2005; Pannese & Procacci, 2002; Ro et al., 1999; Zhou et al., 1999), and the administration of anti-NGF antibodies prevented the hyperalgesia in rats (Cheng et al., 2009; Wild et al., 2007). A recent study, demonstrated the reversion of hyperalgesia after joint mobilization in a model of sciatic nerve crush injury (Martins et al., 2011). In a previous study, from our group, we demonstrated that NM sessions in CCI rats decreases NGF into DRG and reverses the behavioral effects of neuropathic pain, suggesting the involvement of the DRG-expressed NGF with neuropathic
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DOI: 10.3109/08977194.2014.953630
pain and after NM (for revision see Santos et al., 2012) (Martins et al., 2012). Similar results were observed when we analyzed the MPZpositive band in the sciatic nerve. Our results showed an increase of MPZ levels after CCI injury when compared to naive animals and a much higher increase of MPZ after NM treatment (207% above the control). Many studies have demonstrated that MPZ is important for myelin formation and may also play a role in adult axon regeneration (Schweitzer et al., 2003) and mutations of MPZ appear disrupt myelination (Scherer, 1997; Shy, 2006). To determine whether NM accelerated axon outgrowth or the rate of axon regeneration after nerve injury, we evaluated the effect of NM on axon morphology, as determined by electron microscopy. We found that NM helped to improve the myelination axons. Thus, due to trophic (survival- and growth-promoting) effects of NGF (Binder & Scharfman, 2004) and MPZ increased NM appeared to promote the sciatic regeneration by increase of myelinated axons after injury from CCI. These increases coincide with the tissue regeneration (increase of myelin sheath and fibers in animals treated with NM, by transmission electron microscopy). Ankle joint mobilization in rats with neuropathic pain increases sciatic nerve regeneration and inhibits the hyperalgesia, through the possibly inhibition of glial activation in the dorsal horn of the spinal cord (Martins et al., 2011). Our present data are in agreement with those results.
Conclusions In summary, our data reveal that NM sessions are able to improve axonal regeneration in sciatic nerve and its mechanism could be by NGF and MPZ upregulation after a physiotherapy treatment, showing the relevance of a nonpharmacological intervention.
Declaration of interest No competing financial interests exist. This study was supported by FAPESP (2011/22268-0), CNPq, CAPES (Brazil).
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