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Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury
Journal of Clinical Neuroscience xxx (xxxx) xxx
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Experimental study
Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury Ug˘ur Yazar a,⇑, Ertug˘rul Çakır a, Cavit Boz b, Ümit Çobanog˘lu c, Süleyman Baykal a a
Karadeniz Technical University, Faculty of Medicine, Department of Neurosurgery, Trabzon, Turkey Karadeniz Technical University, Faculty of Medicine, Department of Neurology, Trabzon, Turkey c Karadeniz Technical University, Faculty of Medicine, Department of Medical Pathology, Trabzon, Turkey b
a r t i c l e
i n f o
Article history: Received 23 September 2019 Accepted 5 January 2020 Available online xxxx Keywords: Peripheral nerve Melatonin Electroneuromyography Sciatic nerve functional index
a b s t r a c t The aim of this study was to determine the curative effects of high-dose (100 mg/kg) melatonin on peripheral nerve injury. Forty male Wistar albino rats were randomized into four groups as sham, vehicle, melatonin, and ischemia and their right sciatic nerves were exposed. The process was terminated in the sham group. In the other groups, nerve injury was induced by clip compression. The vehicle group was intraperitoneally administered ethanol 0.1 cc (melatonin solvent), while the melatonin group was intraperitoneally administered a single dose of melatonin (100 mg/kg). Following the surgery, sciatic nerve functional index (SFI) was measured using walking track analysis on days 7, 14, and 21, and latency, amplitude, and muscle action potentials (MAP) field values were measured using electroneuromyography (ENMG) on day 21. Histopathologically, edema, axonal degeneration, myelin damage, and inflammatory response were evaluated in all groups. SFI values were noted to be statistically significantly different among the vehicle, melatonin, and ischemia groups, and the melatonin group showed a faster recovery. In the ENMG evaluations, higher amplitude and field values in the melatonin group indicated that melatonin accelerated peripheral nerve recovery. Histopathologically, although fibers with loss of myelin were identified in the melatonin group, the myelin sheath was preserved in general and the axonal structure was noted to be normal. A single injection of high-dose melatonin was found to preserve myelin sheath, prevent axonal loss, and accelerate functional recovery during the nerve regeneration in peripheral nerve injury. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Peripheral nerve injury, which is frequently encountered, may occur due to mechanical factors such as crushing or cutting; it may develop due to ischemic, radial, or chemical factors, and is a significant cause of long-term morbidity. Following the injury, histopathological changes occur in the distal and proximal nerve segments [8,20,21,31]. Since the peripheral nervous system primarily consists of lipids, it is highly sensitive to lipid peroxidation. Additionally, the neural tissue does not include highly active oxidative defense mechanisms. Since lipid peroxidation is potentially harmful, it may alter the fluidity and permeability of neural membranes. The potential damage caused by lipid peroxidation alters the fluidity and permeability of neural membranes and affects the structural integrity and functionality of membranebound receptors and enzymes [35]. Functional recovery following ⇑ Corresponding author. E-mail address: [email protected] (U. Yazar).
peripheral nerve injury is often not good; however, unlike in the central nervous system, regeneration in the peripheral nerves is possible [12]. Depending on the grade and severity of injury, functional remyelination and axonal regeneration are achieved by the reinnervation of sensory receptors or motor end plates. Seddon et al. classified the grade of nerve injuries on the basis of the severity [34]. Being a major neurosecretory product of the pineal gland, melatonin (N-acetyl-5-methoxytryptamine) shows antioxidant [15], geroprotective, and anticarcinogenic [3] properties in biological systems. Melatonin is known to reduce the harmful effects of free radicals in the central nervous system by scavenging free radicals or decreasing the activity of nitric oxide synthase [6,25,30,33]. In addition, reportedly, the early administration of melatonin following traumatic sciatic nerve injury caused by acute blunt injury could decrease lipid peroxidation, axonal damage, and myelin destruction [14,35]. The aim of this study was to determine and discuss the neuroprotective effects of high-dose (100 mg/kg) melatonin on periph-
https://doi.org/10.1016/j.jocn.2020.01.006 0967-5868/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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eral nerve injury induced by the clip compression technique in rats using sciatic functional index, electrophysiological evaluations, and histopathological analyses. 2. Materials and methods 2.1. Animals In this study, 40 healthy male Wistar albino rats weighing 180– 270 g were used. The rats were housed on a 12 h day/night cycle at 20 °C–22 °C (two rats in one cage) and were fed standard laboratory animal feed and water. All experimental protocols were approved by Karadeniz Technical University (K.T.U.), Animal Experiments Local Ethics Committee. The rats were made to fast for approximately 24 h prior to the study, and only water was provided. The experimental animals were randomized into 4 groups, each including 10 animals (Group I: sham; Group II: vehicle; Group III: melatonin, and Group IV: ischemia). 2.2. Induction of peripheral nerve injury or surgical procedures All the rats were anesthetized with 30 mg/kg intraperitoneal administration of ketamine hydrochloride (Ketalar, Pfizer Ilaclari Limited Sirketi, Istanbul, Turkey). The right femoral region was shaved on the operating table in the prone position. The surgical field was sterilized with 10% povidone-iodine solution and the right sciatic nerves were exposed (Fig. 1a). The process was terminated in the sham group. In the vehicle group, each sciatic nerve was compressed using a clip with a compression force of 50 g/ cm (Yasargil FE 693 aneurysm clip-Aesculap) (Fig. 1b). This compression trauma was performed 1 cm proximal to the bifurcation point of the nerve for 150 s and the compression was terminated (Fig. 1c). Next, the distal segment of compressed nerves was marked using a 5/O nylon suture. The layers were properly closed. In the melatonin and ischemia groups, peripheral nerve injury was induced by the same clip compression technique. Immediately after the compression, the vehicle group was intraperitoneally administered ethanol 0.1 cc (melatonin solvent), while the melatonin group was intraperitoneally administered a single dose of melatonin (100 mg/kg). No medication was administered to the ischemia group. No postoperative antibiotics were used. All the rats were kept alive for a period of three weeks. 2.3. The sciatic functional index evaluation Following the procedure, sciatic nerve functional index (SFI) was measured using walking track analysis on days 7, 14, and 21. One end of the walking track (length: 42 cm, width: 8.2 cm, and height: 12 cm) was left open to establish a path to a dark room, wherein the walking track was covered with millimetric paper. After the hind legs of the rats were immersed in drawing ink, they were put on this track (Fig. 2), and the parameters were measured to be used in SFI calculations (Fig. 2b). SFI was calculated using the following formula [5] in the measured parameters. An SFI value of 0 was considered normal, whereas a SFI of 100 represented complete nerve dysfunction.
SFI ¼ 38:3
ðEPL NPLÞ ðETS NTSÞ ðEIT NIT Þ þ 109:5 þ 13:3 8:8 NPL NTS NIT
2.4. Electrophysiological evaluation Electroneuromyographic (ENMG) measurements were performed on day 21 following the surgery using the Medelec TECA Premiere device. The data were recorded using self-adhesive
Fig. 1. Induction of sciatic nerve injury in rats: a) surgical dissection and exposure, b) crush injury, c) appearances of siatic nerve after removal of the compression clip.
modified electrodes (3 3 mm). The active electrode was placed on the peroneal muscle at 1.5 cm distal of the clip compression line, whereas the reference electrode was placed on the distal point of muscle around the ankle. For stimulation, monopolar needle electrodes were used. Low-intensity electrical stimulation (duration = 0.1 ms, voltage < 10 mV) was provided through the sciatic notch using the active needle electrode to achieve supramaximal stimulation in the nerves. The latency was considered as the time to the first wave in the stimulation artifact, whereas the amplitude was considered as the voltage from the isoelectric point to the negative peak. The field was considered as the wave field between the isoelectric line and negative peak. During the recording, the device filter was set to 20 Hz–3 kHz, sweep speed to 2 ms/division, and sensitivity to 1–10 mV/division. As the criteria in the
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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Fig. 2. a) The records of rat’s footprints. b) Calculated parameters of SFI. EPL, experimental print length; NPL, normal print length; ETS, experimental toe spread; NTS, normal toe spread; EIT, experimental intermediate toe spread, NIT, normal intermediate toe spread.
ENMG evaluations, latency, compound muscle action potential amplitude and field were analyzed. 2.5. Histopathological analysis Following the ENMG evaluation, the rats were sacrificed with pentobarbital. The sciatic nerves were removed to position the clip compression line in the middle and include the proximal and distal segments. Next, the sciatic nerve was identified in 10% neutral buffered formalin solution. Then, tissue sampling was performed, obtaining the longitudinal segments passing through the clip compression line; 6–8 mm of segments were obtained from the paraffin-embedded tissues. Following routine follow-up, the preparations were stained with Hematoxylin and Eosin (HE), and then with Luxol fast blue (LB) to reveal the myelin sheath. All segments were evaluated under light microscope. In the histopathological evaluation, edema, axonal degeneration, myelin damage, and inflammatory response were taken into consideration. 2.6. Statistical analysis In the statistical evaluations, obtained analysis results in numbers were evaluated and expressed as mean ± standard deviation of the mean (X ± SD). The Kolmogorov-Smirnov test was used to verify if the data were normally distributed. The temporal variation of the SFI parameters in non-normally distributed data of more than
two dependent groups was evaluated by the Friedman two-way analysis of variance (ANOVA). In case of a significant difference detected in the Friedman two-way analysis of variance, the Wilcoxon test was used to determine the time periods between which this difference was noted. Following the ENMGs, inter-group latency, amplitude and muscle action potential (MAP) area were examined by the Kruskal–Wallis test used for non-normally distributed data of more than two independent groups. The MannWhitney U test was performed for post-hoc comparisons. p < 0.05 was considered statistically significant.
3. Results 3.1. Sciatic functional index evaluation After peripheral nerve injury was induced by clip compression, the recovery was evaluated using sciatic functional index on days 7, 14, and 21 (Table 1 and Fig. 3). The SFI values in all rats before the experiment were close to 0. As there was no sciatic nerve injury in the sham group, the postoperative SFI values were quite favorable. In the other three groups, the postoperative SFI values reached negative values, which indicated the loss of function. The SFI values were increased over time following nerve injury in all groups. The mean SFI values on postoperative days 7, 14, and 21 in the melatonin-administered group were 46.10 ± 11.68, 29.37 ± 9.67 and 14.98 ± 5.04, respectively, and these values
Table 1 SFI scores for the experimental groups (n = 10 for each) in 7th, 14th and 21st day after operation.
Sham Vehicle Melatonin Ischemia p** value
7th day mean ± Sd
14th day mean ± Sd
21st day mean ± Sd
p* value
16.29 50.41 46.10 53.93 0.0001
14.42 37.93 29.37 40.42 0.0001
6.42 ± 4.86 25.17 ± 3.90 14.98 ± 5.04 28.49 ± 4.55 0.0001
0.001 0.0001 0.0001 0.0001
± ± ± ±
3.895 5.01 11.68 7.06
±±3.383 ± 6.43 ± 9.67 ± 6.72
Data are presented as the mean ± standard deviation (X ± SD). p < 0.05. * Evaluation of DT in each group was analyzed using the Friedman Test. ** Group comparisons was employed using the Kruskal-Wallis Test.
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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the second weeks (p = 0.01) and between the third weeks (p = 0.0001). On the basis of these data, it was observed that the functional recovery increased over time following sciatic nerve injury, and the recovery was more rapid particularly in the melatonin group (Fig. 3). 3.2. Electrophysiological evaluation The latency, amplitude, and muscle action potential (MAP) field values were measured using electroneuromyography (ENMG) on day 21 following the initiation of the study (Table 2 and Fig. 4). The mean latency values of the groups are presented in Fig. 4a. Accordingly, the most rapid latency was in sham, melatonin, ischemia and vehicle groups, respectively. The comparison between all groups (Table 2) revealed no significant difference in latency values (p = 0.006) and a significant difference in amplitude (p = 0.0001, Fig. 4b) and MAP (p = 0.0001, Fig. 4c) values. The paired comparisons for latency, amplitude, and MAP field values are presented in Table 3. Accordingly, in the paired comparison of latency values, no statistically significant difference was noted between the groups except for the sham–vehicle group (p = 0.001) (Table 3, Fig. 4). The examination of amplitude values revealed a significant difference between all the groups except for the vehicle-ischemia group (p = 0.496). Although no statistically significant difference was noted between the sham–melatonin (p = 0.130) and vehicle–ischemia (p = 0.596) groups in terms of MAP field values, a statistically significant difference was found in the other groups (Table 3). 3.3. Light microscopic evaluation
Fig. 3. Comparisons of sciatic function index (SFI) for the groups on a) 7th, b) 14th, c) 21st day after operation. The values are means ± standard deviation (X ± SD) for 10 rats for each group.
For determining the recovery effect of melatonin in peripheral nerve injury, a histomorphological analysis was performed in the distal end of crushed sciatic nerve of rats on postoperative day 21. In the examination of the segments stained with HE, no pathological findings were identified in the distal segment of peripheral nerve in the sham group, whereas edema, mononuclear inflammatory cell proliferation, and degeneration were detected in the vehicle, melatonin, and ischemia groups (Fig. 5). In the examination of the segments stained with LB, no pathological findings were identified in the distal segment of peripheral nerve in the sham group, whereas the fibers with loss of myelin were identified, the myelin sheath was preserved, and the axonal structure was normal in the melatonin group, and the segments were similar to those in the sham group. In the vehicle and ischemia groups, edema, myelin and axon loss, and axonal atrophy were observed (Fig. 6). 4. Discussion
in the ischemia group were 53.93 ± 7.06, 40.42 ± 6.72 and 28. 49 ± 4.55, respectively. The SFI values were statistically significantly different among the vehicle, melatonin, and ischemia groups, and the melatonin group showed a faster recovery than the other groups. The recovery achieved by the melatonin group on postoperative day 14 could be achieved by the ischemia group only on postoperative day 21 (Table 1 and Fig. 3). In the melatonin-administered group, a significant difference was noted in the measurements between the first week and the second week (p = 0.005), between the first week and the third week (p = 0.005), and between the second week and the third week (p = 0.005), and a rapid recovery was observed (Table 1). In addition, the comparison between the melatonin and ischemia groups revealed no statistically significant difference between the first weeks (p = 0.049) and a statistically significant difference between
Due to increasing industrialization, peripheral nerve injury has become one of the most significant and frequently occurring Table 2 The results of electrophysiological measurements for the experimental groups (n = 10 for each).
Sham Vehicle Melatonin Ischemia p
Latency (ms)
Amplitude (mV)
MAP area (mVs)
1.12 ± 1.64 ± 1.38 ± 1.49 ± 0.006
35.50 ± 7.50 10.64 ± 7.39 23.32 ± 5.71 7.94 ± 3.61 0.0001
37.10 ± 15.62 ± 33.66 ± 14.95 ± 0.0001
0.19 0.24 0.27 0.42
6.21 10.03 11.89 10.04
MAP, muscle action potential. Data are recorded as the mean ± standard deviation (X ± SD). p values according to Kruskal-Wallis test. p < 0.05.
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
U. Yazar et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx
Fig. 4. The nerve electrophysiological functions, comprising of a) latency, b) amplitude and muscle action potential (MAP) area of the nerve post-operation. The values are means ± standard deviation (X ± SD) for 10 rats for each group.
Table 3 Statistical results of the electrophysiological measurements in group comparisons. p values at 21st day
Sham-vehicle Sham-melatonin Sham-ischemia Vehicle-melatonin Vehicle-ischemia Melatonin-ischemia
Latency (ms)
Amplitude (mV)
MAP area (mVs)
0.001 0.12 0.30 0.149 0.545 0.649
0.0001 0.003 0.0001 0.003 0.496 0.0001
0.0001 0.130 0.001 0.002 0.596 0.001
MAP, muscle action potential. Group comparisons was employed using the Mann-Whitney U test. p < 0.05.
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injuries in our society [9,11]. The rate of successful nerve repair surgeries for peripheral nerve injuries have significantly increased owing to micro-surgical interventions becoming widespread and advances made in histopathological methods [22]. Permanent disorders in sensory and motor functions may develop even though optimal surgical interventions are performed [10,13], and the recovery results obtained are not sufficient to eliminate the negative effects in the patients’ daily lives [22]. Although the mechanisms of post-traumatic repair in peripheral and central nervous systems are quite different, the primary success of antioxidant agents in repairing central nervous system injuries indicates that their pharmacological uses may be beneficial in repairing peripheral nervous system injuries [1,18,24]. Contemporary pharmacological methods used in the repair of peripheral nervous system injuries include the use of non-steroidal anti-inflammatory drugs, steroids, nerve growth factors, thyroid hormones, growth hormones, adrenocorticotropic hormone, and insulin-like peptides [29,32]. The neuroprotective effects of melatonin have been extensively studied [14,16,19,35–37]. It has been determined in various experimental models that the administration of melatonin following traumatic peripheral nerve injury prevents the formation of neuroma and the accumulation of collagen and, therefore, has positive effects on the number of axons and the thickness of myelin sheath [36]. In a study by Shokouhi et al. [35], the rats were administered melatonin at doses of 10 mg/kg and 50 mg/kg following blunt sciatic nerve injury. They demonstrated that the dose of 50 mg/kg had a potent neuroprotective effect, and the peripheral neuronal fibers were protected from lipid peroxidation in tissue MDA level and transmission electron microscopy studies. In a study by Zencirci et al. [37], sciatic nerve injury was induced by crushing the sciatic nerve with surgical forceps and melatonin was administered at doses of 5 and 10 mg/kg. They indicated in SCI and electrophysiological analyses that the administration of melatonin contributed to the repair of sciatic nerve injury. Although studies have demonstrated the neuroprotective effects of melatonin in nerve injury, additional clinical studies should be performed on patients and the effects of different doses should be investigated. In the studies demonstrating the neuroprotective effects of melatonin in peripheral nerve injury, the doses of 5, 10, 20 and 50 mg/kg/day were generally used [17,35,8]. In a study by Gul et al. [14] wherein a single dose of melatonin (50 and 100 mg/kg) was intraperitoneally administered for repairing post-traumatic spinal cord injury, it was revealed that although melatonin did not show a dosedependent activity on lipid peroxidation in the early period, it had neuroprotective effects on white matter axons and myelin sheaths. However, they indicated that the dose of 100 mg/kg provided statistically better outcomes compared with the dose of 50 mg/kg in the melatonin dose groups. In a study by Shokouhi et al. [35] using low- (10 mg/kg) and high- (50 mg/kg) doses of melatonin following blunt sciatic nerve injury, it was reported that the high dose of melatonin had potential neuroprotective effects and could protect peripheral neural fibers from the harmful effects of lipid peroxidation. Our study investigated the efficacy of highdose melatonin (100 mg/kg) with single administration in peripheral nerve injury induced by clip compression. The compression with an aneurysm clip following the exposure of sciatic nerve may result in a predefined severity of injury, and a varied severity of injury can be induced by changing the clip closure force and compression duration. The clip compression causes ischemia as well as mechanical injury. The primary aim following nerve injury is to achieve functional recovery as soon as possible. One of the methods used to determine functional recovery is walking analysis [7]. SFI is a very useful tool for evaluating the func-
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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Fig. 5. Representative Hematoxylin-eosin (HE; 100) stained images. a) Sham group, revealing no pathological findings. b) Vehicle group, showing mononuclear inflammatory cell proliferation. c, d) Melatonin and ischemia group, respectively, displaying edema and mononuclear inflammatory cell proliferation.
Fig. 6. Representative Luxol fast blue (LB) stained images. a) Sham group, revealing no pathological findings (200). b) Vehicle group, displaying edema, myelin and axon loss, and axonal atrophy (200). c) Melatonin group, consisting of preserved myelin sheaths, normal axonal structures and few fibers with loss of myelin (400). d) Ischemia group, showing edema, myelin and axon loss, and axonal atrophy (200).
tional recovery of nerve [23]. Despite numerous controversies, SFI continues to be used in the evaluation of functional recovery following compression trauma, tensile stress, nerve grafting, and surgical trauma [2]. SFI and walking analysis have proven to be affordable, reproducible, and reliable methods for the evaluation of sciatic nerve injury and its repair [26]. In this study, the temporal variation of SFI in all rats following three weeks of clip compression was examined, and the recovery was observed in all groups over time. When the injury and recovery period were compared, it was found that acute compression with intraperitoneal injection
of melatonin was highly effective in the functional recovery of sciatic nerve and the melatonin group returned to the normal walking pattern more quickly compared with the ischemia and vehicle groups. Increased SFI values indicate the recovery in motor and sensory functions as a result of nerve and muscle regeneration. Various authors have also addressed the higher SFI values in the melatonin groups compared with the non-melatonin groups in peripheral nerve injury [4,29,37]. The evaluation of peripheral nerve regeneration by ENMG provides significant clues on regeneration. The low amplitude
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
U. Yazar et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx
measured in the damaged nerve is particularly a sign of axonal loss and partially demyelination, and additionally indicates demyelination due to low axonal loss and transmission inhibition. It is also known that there is a direct correlation between amplitude and the number of active axons. The latency reflects the fastest axonal conduction. The field and amplitude measurements reflect the nerve fibers with the fastest conduction as well as those with slower conduction. However, those are not reflected in latency measurement [27]. In this study, as the criteria for ENMG evaluation, latency, amplitude, and field values were taken into consideration. In the ENMG results at the end of the third week, the amplitude and field values in the melatonin group were statistically significantly different from those in the ischemia and vehicle groups. These results showed that axonal damage and demyelination in the melatonin group were less compared to that of the ischemia and vehicle groups. Myelin sheaths are rich lipid sources and are the main target of lipid peroxidation that develops due to the influence of free radicals during trauma. Trauma not only damages phospholipids of the neural membranes, but it also makes myelin proteins more susceptible to attacks by reactive oxygen species. Melatonin helps the maturation and development of myelin sheath in regenerated nerve and reduces the damage of myelin sheath and protects it from the effects of peroxidation [35]. In the histopathological evaluation, no difference was observed in the distal segment of compression line between the melatonin-administered group and the ischemia and vehicle groups in the segments stained with HE. However, it was observed in the segments stained with LB that the myelinated fibers were preserved with no pathological images in the axonal structures, whereas the apparent loss of myelin and axon and axonal atrophy were observed in the ischemia and vehicle groups. 5. Conclusion The finding of our study show that a high dose of intraperitoneally administered melatonin (100 mg/kg) following peripheral nerve compression trauma, regardless of its mechanism of action, has positive effects on the regeneration of nerve, generally preserves the myelin sheath, prevents axonal loss, and accelerates the functional recovery. We suggest that melatonin is a useful agent in the treatment of peripheral nerve injury. However, studies exist, including the present study, on the selection of the effective dose of melatonin in the treatment of nerve injury [14,35], and additional studies on the selection of the effective dose, the mechanisms of action, and the side effects of melatonin should be conducted to be able to administer melatonin in clinical practice. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This paper represents a part of the first author PhD thesis under the supervision of the second author. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Al Moutaery K, Arshaduddin M, Tariq M, Al Deeb S. Functional recovery and vitamin E level following sciatic nerve crush injury in normal and diabetic rats. Int J Neurosci 1998;96:245–54.
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Experimental study
Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury Ug˘ur Yazar a,⇑, Ertug˘rul Çakır a, Cavit Boz b, Ümit Çobanog˘lu c, Süleyman Baykal a a
Karadeniz Technical University, Faculty of Medicine, Department of Neurosurgery, Trabzon, Turkey Karadeniz Technical University, Faculty of Medicine, Department of Neurology, Trabzon, Turkey c Karadeniz Technical University, Faculty of Medicine, Department of Medical Pathology, Trabzon, Turkey b
a r t i c l e
i n f o
Article history: Received 23 September 2019 Accepted 5 January 2020 Available online xxxx Keywords: Peripheral nerve Melatonin Electroneuromyography Sciatic nerve functional index
a b s t r a c t The aim of this study was to determine the curative effects of high-dose (100 mg/kg) melatonin on peripheral nerve injury. Forty male Wistar albino rats were randomized into four groups as sham, vehicle, melatonin, and ischemia and their right sciatic nerves were exposed. The process was terminated in the sham group. In the other groups, nerve injury was induced by clip compression. The vehicle group was intraperitoneally administered ethanol 0.1 cc (melatonin solvent), while the melatonin group was intraperitoneally administered a single dose of melatonin (100 mg/kg). Following the surgery, sciatic nerve functional index (SFI) was measured using walking track analysis on days 7, 14, and 21, and latency, amplitude, and muscle action potentials (MAP) field values were measured using electroneuromyography (ENMG) on day 21. Histopathologically, edema, axonal degeneration, myelin damage, and inflammatory response were evaluated in all groups. SFI values were noted to be statistically significantly different among the vehicle, melatonin, and ischemia groups, and the melatonin group showed a faster recovery. In the ENMG evaluations, higher amplitude and field values in the melatonin group indicated that melatonin accelerated peripheral nerve recovery. Histopathologically, although fibers with loss of myelin were identified in the melatonin group, the myelin sheath was preserved in general and the axonal structure was noted to be normal. A single injection of high-dose melatonin was found to preserve myelin sheath, prevent axonal loss, and accelerate functional recovery during the nerve regeneration in peripheral nerve injury. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Peripheral nerve injury, which is frequently encountered, may occur due to mechanical factors such as crushing or cutting; it may develop due to ischemic, radial, or chemical factors, and is a significant cause of long-term morbidity. Following the injury, histopathological changes occur in the distal and proximal nerve segments [8,20,21,31]. Since the peripheral nervous system primarily consists of lipids, it is highly sensitive to lipid peroxidation. Additionally, the neural tissue does not include highly active oxidative defense mechanisms. Since lipid peroxidation is potentially harmful, it may alter the fluidity and permeability of neural membranes. The potential damage caused by lipid peroxidation alters the fluidity and permeability of neural membranes and affects the structural integrity and functionality of membranebound receptors and enzymes [35]. Functional recovery following ⇑ Corresponding author. E-mail address: [email protected] (U. Yazar).
peripheral nerve injury is often not good; however, unlike in the central nervous system, regeneration in the peripheral nerves is possible [12]. Depending on the grade and severity of injury, functional remyelination and axonal regeneration are achieved by the reinnervation of sensory receptors or motor end plates. Seddon et al. classified the grade of nerve injuries on the basis of the severity [34]. Being a major neurosecretory product of the pineal gland, melatonin (N-acetyl-5-methoxytryptamine) shows antioxidant [15], geroprotective, and anticarcinogenic [3] properties in biological systems. Melatonin is known to reduce the harmful effects of free radicals in the central nervous system by scavenging free radicals or decreasing the activity of nitric oxide synthase [6,25,30,33]. In addition, reportedly, the early administration of melatonin following traumatic sciatic nerve injury caused by acute blunt injury could decrease lipid peroxidation, axonal damage, and myelin destruction [14,35]. The aim of this study was to determine and discuss the neuroprotective effects of high-dose (100 mg/kg) melatonin on periph-
https://doi.org/10.1016/j.jocn.2020.01.006 0967-5868/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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eral nerve injury induced by the clip compression technique in rats using sciatic functional index, electrophysiological evaluations, and histopathological analyses. 2. Materials and methods 2.1. Animals In this study, 40 healthy male Wistar albino rats weighing 180– 270 g were used. The rats were housed on a 12 h day/night cycle at 20 °C–22 °C (two rats in one cage) and were fed standard laboratory animal feed and water. All experimental protocols were approved by Karadeniz Technical University (K.T.U.), Animal Experiments Local Ethics Committee. The rats were made to fast for approximately 24 h prior to the study, and only water was provided. The experimental animals were randomized into 4 groups, each including 10 animals (Group I: sham; Group II: vehicle; Group III: melatonin, and Group IV: ischemia). 2.2. Induction of peripheral nerve injury or surgical procedures All the rats were anesthetized with 30 mg/kg intraperitoneal administration of ketamine hydrochloride (Ketalar, Pfizer Ilaclari Limited Sirketi, Istanbul, Turkey). The right femoral region was shaved on the operating table in the prone position. The surgical field was sterilized with 10% povidone-iodine solution and the right sciatic nerves were exposed (Fig. 1a). The process was terminated in the sham group. In the vehicle group, each sciatic nerve was compressed using a clip with a compression force of 50 g/ cm (Yasargil FE 693 aneurysm clip-Aesculap) (Fig. 1b). This compression trauma was performed 1 cm proximal to the bifurcation point of the nerve for 150 s and the compression was terminated (Fig. 1c). Next, the distal segment of compressed nerves was marked using a 5/O nylon suture. The layers were properly closed. In the melatonin and ischemia groups, peripheral nerve injury was induced by the same clip compression technique. Immediately after the compression, the vehicle group was intraperitoneally administered ethanol 0.1 cc (melatonin solvent), while the melatonin group was intraperitoneally administered a single dose of melatonin (100 mg/kg). No medication was administered to the ischemia group. No postoperative antibiotics were used. All the rats were kept alive for a period of three weeks. 2.3. The sciatic functional index evaluation Following the procedure, sciatic nerve functional index (SFI) was measured using walking track analysis on days 7, 14, and 21. One end of the walking track (length: 42 cm, width: 8.2 cm, and height: 12 cm) was left open to establish a path to a dark room, wherein the walking track was covered with millimetric paper. After the hind legs of the rats were immersed in drawing ink, they were put on this track (Fig. 2), and the parameters were measured to be used in SFI calculations (Fig. 2b). SFI was calculated using the following formula [5] in the measured parameters. An SFI value of 0 was considered normal, whereas a SFI of 100 represented complete nerve dysfunction.
SFI ¼ 38:3
ðEPL NPLÞ ðETS NTSÞ ðEIT NIT Þ þ 109:5 þ 13:3 8:8 NPL NTS NIT
2.4. Electrophysiological evaluation Electroneuromyographic (ENMG) measurements were performed on day 21 following the surgery using the Medelec TECA Premiere device. The data were recorded using self-adhesive
Fig. 1. Induction of sciatic nerve injury in rats: a) surgical dissection and exposure, b) crush injury, c) appearances of siatic nerve after removal of the compression clip.
modified electrodes (3 3 mm). The active electrode was placed on the peroneal muscle at 1.5 cm distal of the clip compression line, whereas the reference electrode was placed on the distal point of muscle around the ankle. For stimulation, monopolar needle electrodes were used. Low-intensity electrical stimulation (duration = 0.1 ms, voltage < 10 mV) was provided through the sciatic notch using the active needle electrode to achieve supramaximal stimulation in the nerves. The latency was considered as the time to the first wave in the stimulation artifact, whereas the amplitude was considered as the voltage from the isoelectric point to the negative peak. The field was considered as the wave field between the isoelectric line and negative peak. During the recording, the device filter was set to 20 Hz–3 kHz, sweep speed to 2 ms/division, and sensitivity to 1–10 mV/division. As the criteria in the
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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Fig. 2. a) The records of rat’s footprints. b) Calculated parameters of SFI. EPL, experimental print length; NPL, normal print length; ETS, experimental toe spread; NTS, normal toe spread; EIT, experimental intermediate toe spread, NIT, normal intermediate toe spread.
ENMG evaluations, latency, compound muscle action potential amplitude and field were analyzed. 2.5. Histopathological analysis Following the ENMG evaluation, the rats were sacrificed with pentobarbital. The sciatic nerves were removed to position the clip compression line in the middle and include the proximal and distal segments. Next, the sciatic nerve was identified in 10% neutral buffered formalin solution. Then, tissue sampling was performed, obtaining the longitudinal segments passing through the clip compression line; 6–8 mm of segments were obtained from the paraffin-embedded tissues. Following routine follow-up, the preparations were stained with Hematoxylin and Eosin (HE), and then with Luxol fast blue (LB) to reveal the myelin sheath. All segments were evaluated under light microscope. In the histopathological evaluation, edema, axonal degeneration, myelin damage, and inflammatory response were taken into consideration. 2.6. Statistical analysis In the statistical evaluations, obtained analysis results in numbers were evaluated and expressed as mean ± standard deviation of the mean (X ± SD). The Kolmogorov-Smirnov test was used to verify if the data were normally distributed. The temporal variation of the SFI parameters in non-normally distributed data of more than
two dependent groups was evaluated by the Friedman two-way analysis of variance (ANOVA). In case of a significant difference detected in the Friedman two-way analysis of variance, the Wilcoxon test was used to determine the time periods between which this difference was noted. Following the ENMGs, inter-group latency, amplitude and muscle action potential (MAP) area were examined by the Kruskal–Wallis test used for non-normally distributed data of more than two independent groups. The MannWhitney U test was performed for post-hoc comparisons. p < 0.05 was considered statistically significant.
3. Results 3.1. Sciatic functional index evaluation After peripheral nerve injury was induced by clip compression, the recovery was evaluated using sciatic functional index on days 7, 14, and 21 (Table 1 and Fig. 3). The SFI values in all rats before the experiment were close to 0. As there was no sciatic nerve injury in the sham group, the postoperative SFI values were quite favorable. In the other three groups, the postoperative SFI values reached negative values, which indicated the loss of function. The SFI values were increased over time following nerve injury in all groups. The mean SFI values on postoperative days 7, 14, and 21 in the melatonin-administered group were 46.10 ± 11.68, 29.37 ± 9.67 and 14.98 ± 5.04, respectively, and these values
Table 1 SFI scores for the experimental groups (n = 10 for each) in 7th, 14th and 21st day after operation.
Sham Vehicle Melatonin Ischemia p** value
7th day mean ± Sd
14th day mean ± Sd
21st day mean ± Sd
p* value
16.29 50.41 46.10 53.93 0.0001
14.42 37.93 29.37 40.42 0.0001
6.42 ± 4.86 25.17 ± 3.90 14.98 ± 5.04 28.49 ± 4.55 0.0001
0.001 0.0001 0.0001 0.0001
± ± ± ±
3.895 5.01 11.68 7.06
±±3.383 ± 6.43 ± 9.67 ± 6.72
Data are presented as the mean ± standard deviation (X ± SD). p < 0.05. * Evaluation of DT in each group was analyzed using the Friedman Test. ** Group comparisons was employed using the Kruskal-Wallis Test.
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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the second weeks (p = 0.01) and between the third weeks (p = 0.0001). On the basis of these data, it was observed that the functional recovery increased over time following sciatic nerve injury, and the recovery was more rapid particularly in the melatonin group (Fig. 3). 3.2. Electrophysiological evaluation The latency, amplitude, and muscle action potential (MAP) field values were measured using electroneuromyography (ENMG) on day 21 following the initiation of the study (Table 2 and Fig. 4). The mean latency values of the groups are presented in Fig. 4a. Accordingly, the most rapid latency was in sham, melatonin, ischemia and vehicle groups, respectively. The comparison between all groups (Table 2) revealed no significant difference in latency values (p = 0.006) and a significant difference in amplitude (p = 0.0001, Fig. 4b) and MAP (p = 0.0001, Fig. 4c) values. The paired comparisons for latency, amplitude, and MAP field values are presented in Table 3. Accordingly, in the paired comparison of latency values, no statistically significant difference was noted between the groups except for the sham–vehicle group (p = 0.001) (Table 3, Fig. 4). The examination of amplitude values revealed a significant difference between all the groups except for the vehicle-ischemia group (p = 0.496). Although no statistically significant difference was noted between the sham–melatonin (p = 0.130) and vehicle–ischemia (p = 0.596) groups in terms of MAP field values, a statistically significant difference was found in the other groups (Table 3). 3.3. Light microscopic evaluation
Fig. 3. Comparisons of sciatic function index (SFI) for the groups on a) 7th, b) 14th, c) 21st day after operation. The values are means ± standard deviation (X ± SD) for 10 rats for each group.
For determining the recovery effect of melatonin in peripheral nerve injury, a histomorphological analysis was performed in the distal end of crushed sciatic nerve of rats on postoperative day 21. In the examination of the segments stained with HE, no pathological findings were identified in the distal segment of peripheral nerve in the sham group, whereas edema, mononuclear inflammatory cell proliferation, and degeneration were detected in the vehicle, melatonin, and ischemia groups (Fig. 5). In the examination of the segments stained with LB, no pathological findings were identified in the distal segment of peripheral nerve in the sham group, whereas the fibers with loss of myelin were identified, the myelin sheath was preserved, and the axonal structure was normal in the melatonin group, and the segments were similar to those in the sham group. In the vehicle and ischemia groups, edema, myelin and axon loss, and axonal atrophy were observed (Fig. 6). 4. Discussion
in the ischemia group were 53.93 ± 7.06, 40.42 ± 6.72 and 28. 49 ± 4.55, respectively. The SFI values were statistically significantly different among the vehicle, melatonin, and ischemia groups, and the melatonin group showed a faster recovery than the other groups. The recovery achieved by the melatonin group on postoperative day 14 could be achieved by the ischemia group only on postoperative day 21 (Table 1 and Fig. 3). In the melatonin-administered group, a significant difference was noted in the measurements between the first week and the second week (p = 0.005), between the first week and the third week (p = 0.005), and between the second week and the third week (p = 0.005), and a rapid recovery was observed (Table 1). In addition, the comparison between the melatonin and ischemia groups revealed no statistically significant difference between the first weeks (p = 0.049) and a statistically significant difference between
Due to increasing industrialization, peripheral nerve injury has become one of the most significant and frequently occurring Table 2 The results of electrophysiological measurements for the experimental groups (n = 10 for each).
Sham Vehicle Melatonin Ischemia p
Latency (ms)
Amplitude (mV)
MAP area (mVs)
1.12 ± 1.64 ± 1.38 ± 1.49 ± 0.006
35.50 ± 7.50 10.64 ± 7.39 23.32 ± 5.71 7.94 ± 3.61 0.0001
37.10 ± 15.62 ± 33.66 ± 14.95 ± 0.0001
0.19 0.24 0.27 0.42
6.21 10.03 11.89 10.04
MAP, muscle action potential. Data are recorded as the mean ± standard deviation (X ± SD). p values according to Kruskal-Wallis test. p < 0.05.
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
U. Yazar et al. / Journal of Clinical Neuroscience xxx (xxxx) xxx
Fig. 4. The nerve electrophysiological functions, comprising of a) latency, b) amplitude and muscle action potential (MAP) area of the nerve post-operation. The values are means ± standard deviation (X ± SD) for 10 rats for each group.
Table 3 Statistical results of the electrophysiological measurements in group comparisons. p values at 21st day
Sham-vehicle Sham-melatonin Sham-ischemia Vehicle-melatonin Vehicle-ischemia Melatonin-ischemia
Latency (ms)
Amplitude (mV)
MAP area (mVs)
0.001 0.12 0.30 0.149 0.545 0.649
0.0001 0.003 0.0001 0.003 0.496 0.0001
0.0001 0.130 0.001 0.002 0.596 0.001
MAP, muscle action potential. Group comparisons was employed using the Mann-Whitney U test. p < 0.05.
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injuries in our society [9,11]. The rate of successful nerve repair surgeries for peripheral nerve injuries have significantly increased owing to micro-surgical interventions becoming widespread and advances made in histopathological methods [22]. Permanent disorders in sensory and motor functions may develop even though optimal surgical interventions are performed [10,13], and the recovery results obtained are not sufficient to eliminate the negative effects in the patients’ daily lives [22]. Although the mechanisms of post-traumatic repair in peripheral and central nervous systems are quite different, the primary success of antioxidant agents in repairing central nervous system injuries indicates that their pharmacological uses may be beneficial in repairing peripheral nervous system injuries [1,18,24]. Contemporary pharmacological methods used in the repair of peripheral nervous system injuries include the use of non-steroidal anti-inflammatory drugs, steroids, nerve growth factors, thyroid hormones, growth hormones, adrenocorticotropic hormone, and insulin-like peptides [29,32]. The neuroprotective effects of melatonin have been extensively studied [14,16,19,35–37]. It has been determined in various experimental models that the administration of melatonin following traumatic peripheral nerve injury prevents the formation of neuroma and the accumulation of collagen and, therefore, has positive effects on the number of axons and the thickness of myelin sheath [36]. In a study by Shokouhi et al. [35], the rats were administered melatonin at doses of 10 mg/kg and 50 mg/kg following blunt sciatic nerve injury. They demonstrated that the dose of 50 mg/kg had a potent neuroprotective effect, and the peripheral neuronal fibers were protected from lipid peroxidation in tissue MDA level and transmission electron microscopy studies. In a study by Zencirci et al. [37], sciatic nerve injury was induced by crushing the sciatic nerve with surgical forceps and melatonin was administered at doses of 5 and 10 mg/kg. They indicated in SCI and electrophysiological analyses that the administration of melatonin contributed to the repair of sciatic nerve injury. Although studies have demonstrated the neuroprotective effects of melatonin in nerve injury, additional clinical studies should be performed on patients and the effects of different doses should be investigated. In the studies demonstrating the neuroprotective effects of melatonin in peripheral nerve injury, the doses of 5, 10, 20 and 50 mg/kg/day were generally used [17,35,8]. In a study by Gul et al. [14] wherein a single dose of melatonin (50 and 100 mg/kg) was intraperitoneally administered for repairing post-traumatic spinal cord injury, it was revealed that although melatonin did not show a dosedependent activity on lipid peroxidation in the early period, it had neuroprotective effects on white matter axons and myelin sheaths. However, they indicated that the dose of 100 mg/kg provided statistically better outcomes compared with the dose of 50 mg/kg in the melatonin dose groups. In a study by Shokouhi et al. [35] using low- (10 mg/kg) and high- (50 mg/kg) doses of melatonin following blunt sciatic nerve injury, it was reported that the high dose of melatonin had potential neuroprotective effects and could protect peripheral neural fibers from the harmful effects of lipid peroxidation. Our study investigated the efficacy of highdose melatonin (100 mg/kg) with single administration in peripheral nerve injury induced by clip compression. The compression with an aneurysm clip following the exposure of sciatic nerve may result in a predefined severity of injury, and a varied severity of injury can be induced by changing the clip closure force and compression duration. The clip compression causes ischemia as well as mechanical injury. The primary aim following nerve injury is to achieve functional recovery as soon as possible. One of the methods used to determine functional recovery is walking analysis [7]. SFI is a very useful tool for evaluating the func-
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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Fig. 5. Representative Hematoxylin-eosin (HE; 100) stained images. a) Sham group, revealing no pathological findings. b) Vehicle group, showing mononuclear inflammatory cell proliferation. c, d) Melatonin and ischemia group, respectively, displaying edema and mononuclear inflammatory cell proliferation.
Fig. 6. Representative Luxol fast blue (LB) stained images. a) Sham group, revealing no pathological findings (200). b) Vehicle group, displaying edema, myelin and axon loss, and axonal atrophy (200). c) Melatonin group, consisting of preserved myelin sheaths, normal axonal structures and few fibers with loss of myelin (400). d) Ischemia group, showing edema, myelin and axon loss, and axonal atrophy (200).
tional recovery of nerve [23]. Despite numerous controversies, SFI continues to be used in the evaluation of functional recovery following compression trauma, tensile stress, nerve grafting, and surgical trauma [2]. SFI and walking analysis have proven to be affordable, reproducible, and reliable methods for the evaluation of sciatic nerve injury and its repair [26]. In this study, the temporal variation of SFI in all rats following three weeks of clip compression was examined, and the recovery was observed in all groups over time. When the injury and recovery period were compared, it was found that acute compression with intraperitoneal injection
of melatonin was highly effective in the functional recovery of sciatic nerve and the melatonin group returned to the normal walking pattern more quickly compared with the ischemia and vehicle groups. Increased SFI values indicate the recovery in motor and sensory functions as a result of nerve and muscle regeneration. Various authors have also addressed the higher SFI values in the melatonin groups compared with the non-melatonin groups in peripheral nerve injury [4,29,37]. The evaluation of peripheral nerve regeneration by ENMG provides significant clues on regeneration. The low amplitude
Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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measured in the damaged nerve is particularly a sign of axonal loss and partially demyelination, and additionally indicates demyelination due to low axonal loss and transmission inhibition. It is also known that there is a direct correlation between amplitude and the number of active axons. The latency reflects the fastest axonal conduction. The field and amplitude measurements reflect the nerve fibers with the fastest conduction as well as those with slower conduction. However, those are not reflected in latency measurement [27]. In this study, as the criteria for ENMG evaluation, latency, amplitude, and field values were taken into consideration. In the ENMG results at the end of the third week, the amplitude and field values in the melatonin group were statistically significantly different from those in the ischemia and vehicle groups. These results showed that axonal damage and demyelination in the melatonin group were less compared to that of the ischemia and vehicle groups. Myelin sheaths are rich lipid sources and are the main target of lipid peroxidation that develops due to the influence of free radicals during trauma. Trauma not only damages phospholipids of the neural membranes, but it also makes myelin proteins more susceptible to attacks by reactive oxygen species. Melatonin helps the maturation and development of myelin sheath in regenerated nerve and reduces the damage of myelin sheath and protects it from the effects of peroxidation [35]. In the histopathological evaluation, no difference was observed in the distal segment of compression line between the melatonin-administered group and the ischemia and vehicle groups in the segments stained with HE. However, it was observed in the segments stained with LB that the myelinated fibers were preserved with no pathological images in the axonal structures, whereas the apparent loss of myelin and axon and axonal atrophy were observed in the ischemia and vehicle groups. 5. Conclusion The finding of our study show that a high dose of intraperitoneally administered melatonin (100 mg/kg) following peripheral nerve compression trauma, regardless of its mechanism of action, has positive effects on the regeneration of nerve, generally preserves the myelin sheath, prevents axonal loss, and accelerates the functional recovery. We suggest that melatonin is a useful agent in the treatment of peripheral nerve injury. However, studies exist, including the present study, on the selection of the effective dose of melatonin in the treatment of nerve injury [14,35], and additional studies on the selection of the effective dose, the mechanisms of action, and the side effects of melatonin should be conducted to be able to administer melatonin in clinical practice. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This paper represents a part of the first author PhD thesis under the supervision of the second author. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Al Moutaery K, Arshaduddin M, Tariq M, Al Deeb S. Functional recovery and vitamin E level following sciatic nerve crush injury in normal and diabetic rats. Int J Neurosci 1998;96:245–54.
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Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006
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Please cite this article as: U. Yazar, E. Çakır, C. Boz et al., Electrophysiological, functional and histopathological assessments of high dose melatonin on regeneration after blunt sciatic nerve injury, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.01.006