J. Pineal Res. 2014; 56:322–332

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/jpi.12125

Journal of Pineal Research

Proliferative effects of melatonin on Schwann cells: implication for nerve regeneration following peripheral nerve injury Abstract: Activation of proliferation of Schwann cells is crucial for axonal guidance and successful nerve regeneration following peripheral nerve injury (PNI). Considering melatonin plays an important role in proliferative regulation of central glial cells, the present study determined whether melatonin can effectively promote Schwann cell proliferation and improve nerve regeneration after PNI. The spontaneous immortalized rat Schwann cell line (RSC 96 cells) was first analyzed by quantitative polymerase chain reaction (QPCR) to detect the potential existence of melatonin receptors. The melatonin receptor-mediated signaling responsible for proliferation was examined by measuring the phosphorylation of extracellular signal-regulated kinases (ERK1/2) pathway. The in vivo model of PNI was performed by the end-to-side neurorrhaphy. The quantity of Schwann cells as well as the number of re-innervated motor end plates (MEP) on target muscles was examined to represent the functional recovery of injured nerves. QPCR results indicated that MT1 is the dominant receptor in Schwann cells. Immunoblotting and proliferation assay revealed an enhanced phosphorylation of ERK1/2 and increased number of RSC 96 cells following melatonin administration. Nonselective melatonin receptor antagonist (luzindole) treatment significantly suppressed all the above findings, suggesting that the proliferative effects of melatonin were mediated by a receptordependent pathway. In vivo results corresponded well with in vitro findings in which melatonin effectively increased the amount of proliferated Schwann cells and re-innervated MEP on target muscles following PNI. As melatonin successfully improves nerve regeneration by promoting Schwann cell proliferation, therapeutic use of melatonin may thus serve as a promising strategy to counteract the PNI-induced neuronal disability.

Introduction With the coming of the industrialization, peripheral nerve injury (PNI) has gradually become one of the most common and important injuries in our society [1, 2]. After PNI, the distal stump of injured nerve may undergo Wallerian degeneration which subsequently triggers a cascade of glial cell responses including Schwann cell proliferation and migration [3, 4]. Previous studies have indicated that the Schwann cell retains a high degree of proliferative activity that plays a crucial role in the regenerative process following PNI [5, 6]. Biochemical reports also demonstrated that by clearing the myelin debris, forming the tubular structures, and up-regulating the synthesis of a number of neurotrophic factors, proliferated Schwann cells support the axonal outgrowth and functional recovery [7, 8]. Through serving as a good substrate for the lesioned axons, Schwann cells provide a suitable microenvironment for the maintenance of axo-glial interaction that subsequently promotes successful nerve regeneration [9, 10]. However, the regeneration speed and functional recovery following PNI is generally slow because Schwann 322

Hung-Ming Chang1, Chiung-Hui Liu2, Wen-Ming Hsu3, Li-You Chen4, Han-Pin Wang4, TsungHuan Wu4, Kuan-Ying Chen4, WenHsin Ho4 and Wen-Chieh Liao4,5 1

Department of Anatomy, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 2Graduate Institute of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei, Taiwan; 3Department of Surgery, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan; 4Department of Anatomy, Faculty of Medicine, Chung Shan Medical University, Taichung, Taiwan; 5 Department of Pediatrics, Chung Shan Medical University Hospital, Taichung, Taiwan

Key words: confocal immunofluorescence, endto-side neurorrhaphy, extracellular signalregulated kinases, melatonin, proliferation assay, Schwann cell Address reprint requests to Wen-Chieh Liao, Department of Anatomy, Faculty of Medicine, Chung Shan Medical University, 110, Sec.1, Chien Kuo North Rd., Taichung 402, Taiwan. E-mail: [email protected] Received October 16, 2013; Accepted January 31, 2014.

cells always take much time to proliferate and migrate into the terminal end of lesioned nerves [11]. With regard to this, delivering agents that could effectively promote the migration and activate the proliferation of Schwann cells would not only help us to better understand the molecular mechanisms of axo-glial interactions, but also provide important insights into clinical design of a therapeutic substance that facilitates nerve regeneration following PNI [12]. Melatonin, the chief secretory product of pineal gland, is best known for its effects on circadian rhythmicity, reproductive function, scavenging of free radicals, and antioxidative activity [13–19]. Moreover, the ability to scavenge free radicals extends to melatonin’s metabolites [20]. Within the past few years, the effects of melatonin on cell proliferation or cell survival have been increasingly reported [21–28]. These studies indicate that melatonin stimulates cell proliferation of avian astrocytes [21], dentate neurons [22, 23], epididymal epithelial cells [24], lymphocytes [25, 26], mesenchymal stem cells [27], as well as increases cell survival of hippocampal neurons [28]. Pharmacological studies also reported that melatonin exerts its

Effects of melatonin on Schwann cell proliferation proliferative function on different cell types following a variety of experimental insults [29–32]. It is suggested that the proliferative effects of melatonin are largely mediated through the receptor-dependent signaling pathway [31, 33, 34]. Through phosphorylating the extracellular signal-regulated kinases (ERK1/2), phosphorylated ERK1/2 activates numerous transcriptional factors (such as Elk1) that regulate the downstream proliferative activity [34]. As the activated proliferation of Schwann cells also serves as an important role in the guidance of axonal outgrowth and successful nerve regeneration after PNI [35, 36], exploring the potential effects of melatonin in promoting the Schwann cell proliferation would greatly aid in understanding the beneficial functions of melatonin following PNI, as well as to provide a therapeutic possibility for the clinical use of melatonin in the treatment for PNI-induced functional disability. However, although the effect of melatonin in the enhancement of proliferation on numerous cell types has been well documented, the potential action of melatonin on Schwann cell proliferation has not been reported. Moreover, whether exogenous treatment with melatonin significantly improves nerve regeneration by promoting Schwann cell proliferation following PNI still remains unexplored. Given that the receptor-mediated ERK1/2 signaling is also the essential pathway participating in the regulation of Schwann cell proliferation [37], the present study was first aimed to examine whether melatonin receptors exist on Schwann cells; this was done utilizing the quantitative polymerase chain reaction (QPCR) and immunoblottings. Secondly, in order to evaluate the proliferative effects of melatonin on Schwann cells as well as the subsequent activation of intracellular signaling pathway, the proliferative assay along with the phosphorylated levels of ERK1/2 was quantified on the cultured RSC 96 cells. Finally, as attempts to correlate the in vitro effects of melatonin with the in vivo function, the amount of proliferated Schwann cells in the lesioned nerve together with the number of re-innervated motor end plates (MEP) on the target muscles was further determined under the lesion model of end-to-side neurorrhaphy (ESN). As ESN has previously been reported to take much time to attain successful nerve regeneration than that of end-to-end neurorrhaphy (EEN) or general neurosuture [38], this model was thus selected as a good paradigm for providing enough time for us to evaluate the improving functions of melatonin in the regenerative process following PNI.

Materials and methods Experimental animals Young adult male Wistar rats weighing 200–300 g (n = 30) obtained from the Laboratory Animal Center of the Chung Shan Medical University were used in this study. All experimental animals were housed under the same conditions with controlled temperature and humidity. In the care and handling of all experimental animals, the Guide for the Care and Use of Laboratory Animals (1985) as stated in the United States NIH Guidelines (NIH Publication No. 86-23) was followed. All experimen-

tal procedures with surgical intervention and melatonin administration were also approved by the Laboratory Animal Center Authorities of the Chung Shan Medical University (IACUC Approval No 1760). Surgical procedures and melatonin administration The in vivo model of PNI was performed by means of end-to-side neurorrhaphy (ESN) as described in our previous studies [38–40]. Briefly, after deeply anesthetized with intraperitoneal injection of 7% chloral hydrate (SigmaAldrich, St. Louis, MO, USA), rats were placed on the surgical microscope and an incision was made along the left mid-clavicular line to expose the left brachial plexus. The musculocutaneous nerve (McN) was then transected at the margin of the pectoralis major muscle. Following that, an epineurial window matching the size of McN was slit open on the ulnar nerve (UnN), taking care not to damage its contained axons, so that the cut end of McN could be attached to the UnN (end-to-side). Immediately after ESN, all operated animals were divided into four groups with six animals in each. Group I remained untreated, whereas Group II to IV were received intraperitoneal Ringer’s solution or melatonin administration daily for successive 30 days at the doses of 1 and 10 mg/kg, respectively. An additional six animals without surgical intervention were served as normal control. Melatonin (Sigma-Aldrich) was first dissolved in absolute alcohol and then diluted in Ringer’s solution with final ethanol concentration less than 1%. The potential effects of melatonin on Schwann cell proliferation were evaluated by bromodeoxyuridine (BrdU) immunostaining and S-100b immunoblotting [41, 42]. The anatomical evidence of nerve regeneration was further assessed by detecting the expression of re-innervated motor end plates on target muscles by the use of a-bungarotoxin and PGP 9.5 immunohistochemistry. Perfusion and tissue preparation For mitotic labeling, all experimental animals were pretreated with BrdU (50 mg/kg, i.p.) (Invitrogen, Carlsbad, CA, USA) 4 hr before perfusion. At the end of the survival period after ESN, half of the experimental animals from all groups were deeply anesthetized with 7% chloral hydrate (0.4 mL/100 g) and subjected to transcardiac perfusion with 100 mL of Ringer’s solution, followed by 45 min of fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. After perfusion, the biceps brachii muscle and the distal end of the repaired nerve were removed and kept in a similar fixative for 2 hr. The tissue block was then immersed in graded concentrations of sucrose buffer (10–30%) for cryoprotection at 4°C overnight. Serial 10- and 40-lm-thick sections of the nerve segment and biceps brachii muscle were cut longitudinally, respectively, with a cryostat (CM3050S; Leica Microsystems, Wetzlar, Germany) on the following day. The nerve sections collected were processed for BrdU and S-100b immunostaining, while the muscle sections collected were processed for PGP 9.5 and a-bungarotoxin immunohistochemistry. For immunoblotting, another half of the 323

Chang et al. experimental animals deeply anesthetized were perfused with Ringer’s solution, and then, the pineal gland and musculocutaneus nerve were quickly removed under a dissecting microscope. The samples were stored at
80°C until use. Immunofluorescence staining For immunofluorescence double staining, muscle sections collected were first placed in the blocking medium containing 0.1% Triton X-100, 3% normal goat serum, and 2% bovine serum albumin (all from Sigma-Aldrich) for 1 hr to block nonspecific binding. After several washes in phosphate-buffered saline (PBS), the sections were incubated in rabbit polyclonal PGP 9.5 antibody (1:1000; Chemicon, Temecula, CA, USA) as well as Alexa Fluor 488-conjugated-a-bungarotoxin (1:1000; Molecular Probes, Eugene, OR, USA) with the blocking medium for 24 hr at 4°C. After incubation in primary antibodies, the sections were further incubated with Cy3-conjugated anti-rabbit IgG (1:400; Jackson Immuno-Research, West Grove, PA, USA) to visualize nerve terminals. For Schwann cell proliferation assay, antibody detection of BrdU incorporated into DNA requires pretreatment with hydrogen chloride to expose the BrdU epitope. The nerve sections were then reacted with Alexa Fluor 555-conjugated-BrdU-mouse monoclonal antibody (1:1000; Invitrogen) as well as rabbit polyclonal anti-S-100b antibody (1:400; DAKO, Carpinteria, CA, USA) at 4°C overnight. After incubation in primary antibodies, the sections were further incubated with FITCconjugated anti-rabbit IgG (1:200; Vector, Burlingame, CA, USA) to visualize S-100b immunoreactive Schwann cells. All mounted sections were examined and photomicrographed under the confocal fluorescence microscope (SP5; Leica Microsystems).

8) into an orange-color formazan product. Briefly, the RSC 96 cells were firstly seeded into the 96-well plates (10 lL/well) and incubated in 100 lL of culture medium supplemented with 5% FBS and different concentrations of melatonin. After 24, 48, and 72 hr of incubation, the extents of cell proliferation were determined by using the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich) assay for 4 hr at 37°C. The absorbance at 450 nm was measured to determine the number of viable cells in each well. RNA isolation and quantitative real-time polymerase chain reaction The total RNA was extracted from the cultured cells using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (QPCR) was performed by using the Mx3000P instrument (Stratagene, Santa Clara, CA, USA), and the signal was detected with the Brilliant SYBR Green QPCR Master Mix (Stratagene). The primers used for QPCR were as follows: MT1 (Rattus norvegicus) forward primer 50 -CG GCGGGGAGGAAATAAGAT-30 and reverse primer 50 -TATAGACGTCAGCGCCAAGG-30 ; MT2 (Rattus norvegicus) forward primer 50 -AGA CAGCCAGCACCCAATAC-30 and reverse primer 50 -GC CCAGCATATGGCG AAAAC-30 ; b-actin (Rattus norvegicus) forward primer 50 -CTTCCAGCCTTCCTTCC TGG-30 and reverse primer 50 -GAGCCACCAATCCAC ACAGA-30 . Relative mRNA levels were calculated based on the cycle threshold values, corrected for b-actin, and according to the equation of 2
DCt (DCt = Ct [MTR]
Ct [b-actin]). All experiments were performed in triplicate with pineal gland serving as positive controls [44].

RSC 96 cell culture and treatments For the in vitro cell biology study, the rat Schwann cell line, RSC 96 cells purchased from the Cell Bank, Chinese Academy of Sciences, was grown in DMEM containing 2 mM glutamine, 100 units/mL penicillin, 100 lg/mL streptomycin, and 5% fetal bovine serum (FBS) in 5% CO2 at 37°C. Different levels of melatonin (at the concentrations of 0.1, 1, and 10 nM) dissolved in ethanol were added to the plating medium to test the potential proliferative effects of melatonin on Schwann cells. In order to examine whether the proliferative effects of melatonin were mediated through receptor-dependent pathway, the nonselective melatonin receptor antagonist (luzindole) (Toronto Research Chemical, North York, ON, Canada) at the concentration of 50 lM was added to the culture 1 hr before melatonin administration [43]. Cells received 0.001% ethanol without any level of melatonin were used as vehicle controls. After incubation, the cells were harvested and extracted for further analysis. Cell counting kit-8 (CCK-8) assay The cell viability was estimated using a colorimetric assay based on the conversion of tetrazolium salt (WST324

Fig. 1. Histogram showing the relative mRNA expression of melatonin receptors in RSC 96 cells, pineal gland, and musculocutaneous nerve (McN) by quantitative real-time polymerase chain reaction (QPCR). The signals were normalized to b-actin and analyzed by using the MxPro Software. Note that in both RSC 96 cells and McN, the mRNA level of MT1 was significantly higher than that of MT2, which suggests that MT1 is the major type of receptor participated in the proliferative regulation of Schwann cells.

Effects of melatonin on Schwann cell proliferation Western blotting Both in vivo tissue samples and RSC 96 cells were subjected to Western blot analysis [40]. Briefly, the pineal gland and musculocutaneous nerve removed from experimental animals were firstly homogenized with Kaplan buf-

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fer (50 mM Tris buffer, pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP40, and protease inhibitor cocktail) and clarified by centrifugation. Following that, equal amounts of solubilized proteins and cell lysates were separated on SDS-PAGE (13%) and electroblotted onto a nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were then blocked with 5% skim milk and probed with antibodies against b-actin (1:10000; GeneTex, Irvine, CA, USA), MT1 (1:1000; Abbiotech, San Diego, CA, USA), S-100b (1:1000; DAKO), p-ERK1/ 2, and ERK1/2 (1:1000, both from Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. After incubation with primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) at a dilution of 1:10000 for 1 hr at room temperature. The immunoreaction was visualized with ECL solution (Millipore, Temecula, CA, USA) followed by 2 min of film exposure. BrdU labeling and DNA staining The proliferation of RSC 96 cells was analyzed by cellular mitotic labeling with BrdU [41]. RSC 96 cells pretreated with 50 lM of BrdU for 1 hr were fixed with 4% paraformaldehyde for 15 min and then treated with 2N HCl for 20 min at 37°C. After several washes in PBS, RSC 96 cells were permeabilized with 0.1% Triton X-100, followed by BrdU labeling with Alexa Fluor 555-conjugated-BrdUmouse monoclonal antibody (1:1000; Invitrogen). Cell nuclei were then subjected to DNA staining with Hoechst 33342 (Molecular Probes).

Fig. 2. Immunoblotting (A) and histogram (B) showing the MT1 protein expression in pineal gland, musculocutaneous nerve (McN), and RSC 96 cells. Note that a single band of approximately 38 kDa on immunoblots that corresponded to the molecular weight of MT1 was detected in both pineal gland and McN as well as in RSC 96 cell lysate (A). Quantification of relative density normalized to b-actin showed that MT1 expression was comparatively higher in McN and RSC 96 cells as compared to that of pineal gland which served as an internal positive control (B). *P < 0.05 as compared to the value of pineal gland.

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Statistical analysis All experimental data acquired from in vivo and in vitro studies were firstly subjected to the Kolmogorov–Smirnov test for analyzing the pattern of normality. Those qualified (P > 0.1) were subsequently processed for Student’s t-test or one-way ANOVA followed by Bonferroni post hoc test. The statistical significance was considered if P < 0.05.

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Fig. 3. Line charts showing the potential effects of melatonin (A) and melatonin receptor antagonist (luzindole) (B) on cell viability of RSC 96 cells. The cell viability was estimated by measuring the absorbance at 450 nm for the cell counting kit-8 (CCK-8) assay. Note that following 48 hr of melatonin exposure, the absorbance of CCK-8 was significantly increased with the maximal change observed in the concentration of 1.0 nM of melatonin (A). Also note that in cells pre-incubated with luzindole, the extent of cell viability was significantly suppressed, which suggested that the proliferative effects of melatonin were mediated by receptor-dependent pathway (B). *P < 0.05 as compared to that of vehicle group.

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Fig. 4. Confocal photomicrographs (A) and histogram (B) showing the effects of melatonin on proliferative function of RSC 96 cells. The RSC 96 cell proliferation was determined by BrdU staining (red) and counterstained with Hoechst 33342 (blue). Note that melatonin significantly promoted cell proliferation by increasing the number of BrdU and Hoechst 33342 double-labeled cells (A). The proliferative effect of melatonin was significantly blocked by luzindole in which the number of BrdU immunoreactive cells was significantly decreased (A). Quantitative analysis also revealed that the percentage of BrdU and Hoechst 33342 double-labeled cells was much higher in 1 nM melatonin-treated group as compared to that of vehicle and luzindole-treated ones (B). *P < 0.05 as compared to that of control value. Scale bar = 10 lm.

Results To determine whether melatonin receptor(s) would actually present in peripheral nerve tissue or in Schwann cells, the QPCR and Western blot analysis are performed to detect the related mRNA and protein expression in both

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Fig. 5. Immunoblotting (A) and histogram (B) showing the MT1 protein expression in RSC 96 cells with or without melatonin treatment. Note that melatonin treatment effectively activated the MT1 expression in RSC 96 cells, which suggested that the beneficial effects of melatonin may exert via the MT1-dependent pathway (A). Densitometric analysis also showed an increased expression of MT1 in melatonin-treated group as compared to that of control value (B). *P < 0.05 as compared to that of control group.

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McN and RSC 96 cells. Results from QPCR indicated that mRNA of both melatonin receptors (MT1 and MT2) are expressed in both McN and RSC 96 cells (Fig. 1). The mRNA level of MT1 in RSC 96 cells was much higher than that of MT2, which suggests that MT1 may play a major role in the proliferative regulation of Schwann cells (Fig. 1). In addition, the existence of melatonin receptor in Schwann cell was also proved by Western blotting in which a single band of approximately 38 kDa that corresponded to the molecular weight of MT1 was observed in McN and RSC 96 cells (Fig. 2A). Quantitative analysis revealed that the expression level of MT1 was 1.24-fold and 1.76-fold higher than that of pineal gland in McN and RSC 96 cells, respectively (Fig. 2B). Besides, as attempts to determine the functional role of melatonin in Schwann cells, the cell number and proliferative assay were performed on RSC 96 cells by using the cell counting kit (CCK-8) and BrdU staining method. Results from CCK-8 assay revealed that with the prolongation of the cultural time, the number of RSC 96 cells (as determined by the optical density of the absorbance) was gradually increased (Fig. 3). Following 48–72 hr of incubation, a significant increase in cell viability was detected in melatonin-treated cells in which the maximal increase was observed in cells incubated with 1 nM of melatonin (Fig. 3A). These data indicate that 1 nM of melatonin may have the optimal effect on promoting cell growth, which is therefore suitable to be used for performing the subsequent proliferative analysis. On the other hand, the proliferative effect of melatonin on RSC 96 cells was completely abolished by luzindole, which suggested that the beneficial effect of melatonin was mediated through receptor-dependent pathway (Fig. 3B). The results of BrdU staining corresponded well with the assay of cell viability in which the percentage of proliferative cells was much higher in 1 nM of melatonin-treated group as compared to that of vehicle and luzindole-treated ones (Fig. 4A). Quantitative analysis revealed that the

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Fig. 6. Immunoblotting (A) and histogram (B) showing the effects of melatonin and luzindole on the protein level of total ERK1/2 (t-ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) in RSC 96 cells. Note that melatonin significantly induced the phosphorylation of ERK1/2 in RSC 96 cells (A). However, the protein level of p-ERK1/2 was effectively suppressed by luzindole, which suggested that MT1dependent ERK1/2 pathway is the crucial signaling pathway participating in the regulation of Schwann cell proliferation (A). Densitometric analysis also indicated a relatively higher ratio of p-ERK1/2 to t-ERK1/2 in melatonin-treated groups as compared to that of vehicle or luzindole-treated ones (B). *P < 0.05 as compared to that of control value.

percentage of BrdU and Hoechst 33342 double-labeled cells was estimated to be 18.1  0.3% in 1 nM of melatonin-treated group (Fig. 4B). Correspondingly, the proliferation of RSC 96 cells was significantly blocked by luzindole in which only 8.7  0.1% of RSC 96 cells were double-labeled with BrdU and Hoechst 33342 (Fig. 4B). As the existence of melatonin receptor (Figs 1 and 2) and the proliferative effect of melatonin on Schwann cells (Figs 3 and 4) have been clearly documented, the present study further determined whether the positive effects of melatonin were mediated by MT1 receptor-dependent ERK1/2 pathway. Western blot analysis revealed that following 30 min of melatonin incubation, both MT1 and phosphorylated ERK1/2 (p-ERK1/2) expressions were significantly increased in RSC 96 cells (Figs 5A and 6A). The increased level of MT1 induced by melatonin was quantified to be 1.5-fold higher than that of control level (Fig. 5B). Similar finding was observed in p-ERK1/2 expression in which the ratio of p-ERK1/2 to total ERK1/ 2 (t-ERK1/2) was considerably higher in melatonin-treated group (Fig. 6B). These results indicate that melatonin may

exert its function by binding to MT1 receptor, activating the intracellular ERK1/2 pathway, and ultimately regulating the proliferative activity of Schwann cells. As attempts to determine the functional significance of melatonin in the in vivo condition, the extent of cell proliferation (as demonstrated by BrdU labeling) and the amount of Schwann cells (as revealed by S-100b immunohistochemistry) were further examined in the recipient nerve following ESN. The present results indicated that at 1 month after ESN, the number of proliferated Schwann cells (i.e., BrdU and S-100b double-labeled cells) was gradually increased in the repaired site of the recipient nerve (Fig. 7). Confocal photomicrography estimation revealed that about 224  25% per 500 lm2 of BrdU and S-100b double-labeled cells were detected at the repaired site 1 month later following ESN (Fig. 7B). However, in animals subjected to ESN and then received different doses of melatonin treatment, the number of proliferated Schwann cells was significantly increased (Fig. 7A). Quantitative analysis revealed that the number of BrdU and S-100b double-labeled cells was estimated to be 266  18% and (B)

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Fig. 7. Confocal photomicrographs (A) and histogram (B) showing the effects of melatonin on Schwann cell proliferation 1 month later following end-to-side neurorrhaphy (ESN). Note that in normal rats, numerous Schwann cells [labeled with S-100b (green)] with some proliferative activity [stained with BrdU (red)] were detected in the intact musculocutaneous nerve (McN) (A). However, following ESN, both the number of Schwann cells and the expression of proliferation were remarkably decreased in the repaired McN (A). Also note that in animals subjected to ESN and received different doses of melatonin, the number of proliferative Schwann cells (i.e., doubled-labeled with S-100b and BrdU) was significantly increased with the relatively higher number detected in the group of 1 mg/kg of melatonin (B). Scale bar = 50 lm. *P < 0.05 as compared to that of saline-treated group.

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rescence) along with the re-innervated motor end plates (MEP) on the target muscles (as revealed by a-bungarotoxin staining) was further examined. Results from normal untreated rats indicated that there are nearly 12  3 clusters of MEP innervated by large PGP 9.5-positive [PGP 9.5-(+)] nerve fibers that were detected in the biceps brachii muscle (Fig. 9). Following ESN, both the numbers of innervated MEP (1.5  0.5) and intramuscular PGP 9.5(+) nerve fibers were drastically decreased (Fig. 9). However, in animals subjected to ESN and received different doses of melatonin, most of the MEP were re-innervated by numerous PGP 9.5-(+) nerve fibers with relatively large diameters (6.4  3.8 and 5.1  2.4 in 1 and 10 mg/kg melatonin treatment group, respectively) (Fig. 9). These data thus coincide well with the in vitro findings in which the proliferative effects of melatonin on Schwann cells would greatly contribute to improve the nerve regeneration following peripheral nerve injury.

Discussion

Fig. 8. Immunoblotting (A) and histogram (B) showing the effects of melatonin on the protein level of MT1 and S-100b in the intact and repaired musculocutaneous nerve (McN) following end-toside neurorrhaphy (ESN). Note that ESN extensively reduced MT1 and S-100b expression in the repaired McN (A). However, in animals subjected to ESN and received different doses of melatonin, both the MT1 and S-100b activities were significantly increased (A). Quantitative analysis revealed that the results were somewhat better in the group given 1 mg/kg of melatonin (B). *P < 0.05 as compared to that of saline-treated value.

259  13% per 500 lm2 in 1 mg/kg and 10 mg/kg of melatonin treatment group, respectively (Fig. 7B). The proliferative effect of melatonin on Schwann cells was further verified by immunoblotting in which melatonin administration induced an enhanced expression of S-100b in the injured nerve following ESN (Fig. 8). The beneficial function of melatonin after ESN was also exerted through membrane receptor wherein the MT1 activity was significantly increased following different doses of melatonin treatment (Fig. 8). As the functional significance of melatonin on Schwann cell proliferation is to promote successful nerve regeneration, the number of regenerated nerves (as revealed by PGP 9.5 immunofluo328

The present study has provided the first functional and anatomical evidence that melatonin treatment successfully promotes Schwann cell proliferation (Figs 3, 4, 7 and 8) and improves nerve regeneration following PNI (Fig. 9). The proliferative effect of melatonin on Schwann cells has further been demonstrated to be mediated by MT1 receptor-dependent ERK1/2 pathway (Figs 5 and 6). It is indicated that upon nerve injury, Schwann cells in the distal stump would loss contact with the degenerated axons [35]. Detached Schwann cells would undergo dedifferentiation followed by a series of proliferation [35]. The proliferated Schwann cells would secrete a variety of trophic factors and form bands of B€ unger, which generates a permissive environment for axon sprouting and subsequently regulates the regeneration processes [35]. By serving as a ‘transient target’ for the lesioned nerve, proliferated Schwann cells guide the axonal outgrowth and facilitate the reinnervation of target muscles following PNI [45]. Thus, endogenous activation or exogenous treatment with a specific agent that effectively promotes Schwann cell proliferation would have great potential for clinical use as a therapeutic strategy to counteract the PNI-induced functional deficiency. Herein, we have first demonstrated that melatonin significantly stimulates Schwann cell proliferation in both in vitro and in vivo conditions. The increased expression of Schwann cells induced by melatonin was clearly expressed by cell counting and the proliferative assay (Figs 3 and 4) in RSC 96 cells as well as by quantitative BrdU and S-100b immunoexpressions in the lesioned nerves (Figs 7 and 8). Because the activated proliferation of Schwann cells is highly essential for successful nerve regeneration, the present study further demonstrated that the proliferated effects of melatonin on Schwann cells are positively correlated with the improved nerve recovery. Functional anatomical evidence revealed that the number of re-innervated motor end plates (as labeled by PGP 9.5 and a-bungarotoxin) on target muscles following PNI was much higher in animals receiving melatonin treatment when compared to that of saline-treated ones (Fig. 9). As melatonin distinctly exerts its beneficial effects on improving

Effects of melatonin on Schwann cell proliferation nerve regeneration by successfully promoting the Schwann cell proliferation, our study thus not only innovates a new understanding about the advanced effects of melatonin in peripheral glial cells following PNI, but also sheds important light for the therapeutic use of melatonin in the treatment for PNI-relevant neuronal sequelae. In addition to evaluating the possible effects of melatonin on Schwann cell proliferation, the underlying mechanisms involved in regulating the proliferative function of melatonin on Schwann cells were also examined. The findings indicate that the receptor-mediated ERK1/2 signaling is the essential pathway participating in the proliferative regulation of Schwann cells [37]. Increased ERK1/2 activation would transmit lesioned signals into nucleus for transcriptional regulation of genes related to Schwann cell differentiation and proliferation [46, 47]. Previous studies have indicated that enhanced ERK1/2 expressions were found in regenerating tissues after PNI [48, 49]. Seo et al. [50] also demonstrated that increased ERK1/2 activity would contribute to Schwann cell proliferation and played an important role in axonal regeneration following sciatic nerve injury. It is reported that ERK1/2 could be activated by phosphorylation in response to melatonin [51]. By binding to membranous receptors, melatonin could activate the downstream ERK1/2 pathway and protects neurons from hypoxia or ischemic damage [31, 52]. However, although the regulatory role of melatonin receptor-mediated ERK1/2 activation in the modulation of the neuronal function has been well documented, the possible existence of melatonin receptor in Schwann cells as well as the potential involvement of this pathway in Schwann cell proliferation has not yet been reported. In this regard, the present study has provided for the first time that both MT1 and MT2 melatonin receptors exactly existed in Schwann cells (Figs 1 and 2). Increased expression of MT1 and phosphorylated ERK1/2 was further detected in Schwann cells after melatonin exposure (Figs 5 and 6). The enhanced activation of MT1 and ERK1/2 was correlated well with cell counting and proliferative assay (Figs 3 and 4), which clearly documented that MT1-dependent ERK1/2 signaling is also the major pathway involved in the proliferative regulation of melatonin on Schwann cells. In addition, our QPCR results also detected a

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significantly higher level of MT1 mRNA than that of MT2, which suggested that MT1 is the major type of receptor participated in the proliferative regulation of Schwann cells (Fig. 1). These findings are in good agreement with pharmacological studies in which MT1 has previously been reported to participate in neuronal firing, metabolic function as well as cell proliferation, while MT2 usually takes part in modulating the leukocyte rolling and circadian activity [33, 53, 54]. Nevertheless, caution must be exercised when explaining the neuroprotective function of melatonin because the determination of Schwann cell proliferation and nerve regeneration usually depends on mutual relationship between multiple signaling (such as cAMP, Jak-STAT, Pl3k-Akt, and Wnt/b-catenin pathways) [37, 55]. Thus, extensively elucidating the cross-talk mechanism and potential effects of melatonin on these pathways might provide great insight into the functional significance of melatonin in the management of PNI-relevant functional disability. Aside from triggering the MT1-dependent signaling pathway, one of the most effective actions of melatonin relates to its significant antioxidative and anti-inflammatory activities. Besides directly scavenging free radicals, melatonin could also increase the activity of several antioxidant enzymes as well as suppress the expression of a variety of pro-inflammatory cytokines [17, 56–58]. Previous studies have indicated that enhanced oxidative stress and excessive exposure to pro-inflammatory cytokines following PNI would inhibit Schwann cell differentiation and cause Schwann cell apoptosis [59, 60]. Recent reports also demonstrated that treatment with antioxidants (e.g., quercetin or N-acetylcysteine) or melatonin would increase the Schwann cell activity and facilitate the functional recovery following PNI [61–63]. As melatonin possesses central function on antioxidative and anti-inflammatory regulation, the beneficial effects of melatonin on Schwann cells may also be exerted through its regulatory function on injury-induced cellular processes such as modulating the intracellular oxidative status and suppressing the inflammatory reactions. Another important issue to be addressed is the concentration of melatonin used in this study. Both in vitro and in vivo findings revealed that the promoting effects of melatonin on Schwann cell proliferation was not dose

(C)

(D)

Fig. 9. Confocal photomicrographs showing the neuroregenerative effects of melatonin 1 month later following end-to-side neurorrhaphy (ESN). The expression pattern of re-innervated motor end plates on the target muscle [MEP, as demonstrated by a-bungarotoxin (green) and PGP 9.5 (red)] was used to represent the functional recovery of repaired nerves. It is noted that in normal rats, several MEP innervated by large-diameter nerve fibers (arrows) were identified in the target muscle (A). Following ESN, the number of innervated MEP and the diameter of nerve fibers in the target muscle were drastically decreased (B). However, in animals subjected to ESN and received different doses of melatonin (C, D), the number of re-innervated MEP was significantly increased with a slightly better finding detected in animals receiving 1 mg/kg of melatonin (C). Scale bar = 50 lm.

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Chang et al. dependent because higher levels of melatonin did not cause better results than the lower ones (Figs 3, 6, 7, and 8). In vitro findings of cell counting analysis revealed that after 72 hr of incubation, 1 nM of melatonin, instead of 10 nM, is the most effective concentration in promoting the cell viability (Fig. 3). Results from immunoblottings also showed that the increment of phosphorylated ERK1/2 was most evident in cells treated with 1 nM of melatonin (Fig. 6). Similar findings were also observed in in vivo condition in which the most significant effect of melatonin on Schwann cell proliferation was detected in animals receiving melatonin at the dose of 1 mg/kg, rather than that of 10 mg/kg (Figs 7 and 8). These results are thus in good agreement with several previous studies in which higher levels of melatonin declined the stimulatory effects of cell proliferation when compared with the lower concentration [31, 64, 65]. Although the detailed mechanisms for the difference in proliferative effects of melatonin under different concentrations are not clear, the cross-reaction between different doses of melatonin with other signaling pathway (e.g., acts via not only the MT1, but also directly on other targets including protein kinase C or mitochondria) that triggers variant result may serve as the possible factor contributing to this event [64, 65]. In summary, the present study illustrates for the first time that melatonin improves nerve regeneration by effectively promoting Schwann cell proliferation following peripheral nerve injury. The proliferative effects of melatonin on Schwann cells are mediated through the MT1dependent ERK1/2 pathway. Although the detailed mechanisms underlying the regenerative processes after PNI are still not fully understood, the significant proliferative effect of melatonin on Schwann cells makes this indole an attractive target for clinical use as a therapeutic agent to facilitate axonal regeneration and improve neuronal function following PNI.

Acknowledgements This study is supported in part by the research grants (NSC 99-2320-B-038-019-MY3 and NSC 102-2320-B-040-005) to Dr. H-M Chang and Dr. W-C Liao from the National Science Council, Taiwan. The authors confirm that there are no known conflict of interests associated with this research and there has been no significant financial support for this work that could have influenced its outcome.

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