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The Effects of Exogenous Melatonin on Peripheral Nerve Regeneration and Collagen Formation in Rats
Journal of Surgical Research 166, 330–336 (2011) doi:10.1016/j.jss.2009.06.002
The Effects of Exogenous Melatonin on Peripheral Nerve Regeneration and Collagen Formation in Rats Bekir Atik, M.D.,* Ibrahim Erkutlu, M.D.,† Mustafa Tercan, M.D.,k Hakan Buyukhatipoglu, M.D.,{,1 Mehmet Bekerecioglu, M.D.,‡ and Sadrettin Pence, M.D., Ph.D.§ *Department of Plastic and Reconstructive Surgery, Yuzuncu Yil University, Medical Faculty, Van, Turkey; †Department of Neurosurgery; ‡Department of Plastic and Reconstructive Surgery; §Department of Physiology, Medical Faculty, Gaziantep University, Gaziantep, Turkey; kDepartment of Plastic and Reconstructive Surgery, Haydarpasa Numune Hospital, Istanbul, Turkey; and {Department of Internal Medicine, Harran University School of Medicine, Sanliurfa, Turkey Submitted for publication March 16, 2009
Background. Peripheral nerve damage that requires surgical repair does not result in complete recovery because of collagen scar formation, ischemia, free oxygen radical damage, and other factors. To date, the best treatment method has not yet been determined. In this study, we designed an experimental peripheral nerve injury model, and researched the possible effects of melatonin hormone, based on evidence of its strong antioxidant and cell-protective effects via mimicking the effects of calcium channel blockers. Materials and Methods. We randomized 24 healthy female albino rats into three groups: the pinealectomy group, melatonin group, and control group. In the pinealectomy group, craniotomy, pinealectomy, sciatic nerve transection, and coaptation were performed, and 0.9% NaCl was injected intraperitoneally. In the melatonin group, craniotomy (without pinealectomy), sciatic nerve dissection, and coaptation were performed, and melatonin was injected intraperitoneally, instead of NaCl. In the control group, craniotomy (without pinealectomy), sciatic nerve dissection and coaptation, and intraperitoneal NaCl injection were performed. In each group, nerve recovery was evaluated histologically, functionally, and electrophysiologically. Functional and electrophysiologic evaluations were conducted before surgery and at 4 and 12 wk. Results. At 4 wk, no significant difference was observed between the groups. However, at 12 wk, significant electrophysiologic and functional improvement was observed only in the melatonin group.
1 To whom correspondence and reprint requests should be addressed at Harran University School of Medicine, Gazimuhtarpasa bul. Kalyon is merkezi No.17-18 Sehitkamil, Gaziantep, Turkey. E-mail: [email protected], [email protected].
0022-4804/$36.00 Ó 2011 Elsevier Inc. All rights reserved.
Conclusions. Melatonin seems to have a beneficial effect on nerve recovery. However, this effect is not effective at physiologic doses. Future comparative studies with melatonin versus other nerve-regenerating agents are necessary to determine the clinical utility of melatonin hormone. Ó 2011 Elsevier Inc. All rights reserved. Key Words: peripheral nerve injury; regeneration; melatonin; collagen formation.
INTRODUCTION
Peripheral nerves are the most important structures for providing motor and sensory function in mammalians. Damage to peripheral nerves results in partial or complete loss of these functions. Currently, peripheral nerve injuries and repairs continue to be a major problem. Surgical reconstruction procedures do not yield complete recovery. Many studies have been performed on surgical techniques; however, studies on medications that might positively affect peripheral nerve regeneration are rare [1–4]. When damage occurs to a peripheral nerve, both ischemic and inflammatory processes begin. As part of this process, free oxygen radicals and many toxic agents accumulate around the site of injury. As a result, membrane permeability is disturbed and intracellular calcium influx is stimulated. Intracellular calcium influx, in turn, activates proteolytic pathways, resulting in cell destruction, which include the destruction of neurofilaments and microtubules. Another important point relevant to the regenerative process is that scar tissue forms around the regeneration field. Any scar tissue
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that forms between the proximal and distal ends of a repaired nerve can physically impede the forward development of the axon [3, 5–7]. In this study, we researched the role of the hormone melatonin in the regenerative process. Melatonin is secreted by the pineal gland that lies at the base of the brain. Considerable evidence exists to document the many different roles of melatonin in the human body, including its importance in circadian rhythms, sleep physiology, mental status, reproduction, tumor development, aging, and many other physiologic processes. Its many functions in the human body have not yet been completely understood [8–10]. Many in vivo and in vitro studies have demonstrated that melatonin is a strong free radical removal agent. Melatonin can protect macromolecules, especially DNA, from oxidative damage. It exerts this effect directly, independent of receptors [11–13]. In order to research melatonin’s role in the nerve regeneration process, we designed an experimental peripheral nerve injury model.
Surgical Techniques Rats were starved for 24 h prior to surgery. Anesthesia was induced using xylocaine (Rhompun, 2%; Bayer Drug Industry, Istanbul, Turkey) 10 mg/kg and ketamine hydrochloride (Katalar, 5%; Eczacıbas xı Drug Industry with permission from Parke-Davis, Ann Arbor, MI), which were mixed and given intraperitoneally. Microsurgical techniques were utilized under sterile conditions, using 4 3 4 magnification surgery loops.
Pinealectomy Anesthetized rats were affixed to the operation table at each of their four extremities, as described by Pohlymeyer et al. [14]. Heads and necks were cleaned gently with polyvinylpyrolidon iodine (polyod 10% solution; Drogsan Drug Industry, Ankara, Turkey) and covered with a sterile surgical drape. A 1.5 cm incision was made over the hairless scalp, just behind the eyes. Through this incision, the pineal gland was carefully removed.
Sciatic Nerve Coaptation With the rat in the same position, the sacrum and left lower extremities were shaved and cleaned with polyvinylpyrolidon. Under surgical conditions, a skin incision was made parallel to the long extremity axis. The gluteus maximus muscle was dissected from the femoral bone edges, and the sciatic nerve exposed. The nerve was freed from surrounding tissues. The tibial and peroneal branches were bluntly dissected from each other. Next, the nerve was dissected 15 mm cephalad to the sural, peroneal, and tibial bifurcations. Dissected nerve ends were coapted with prolene suture material.
MATERIAL AND METHODS Study Design and Groups This study was approved by the local ethics committee of Gaziantep University School of Medicine. In this study, we used 24 healthy female albino rats, each weighing 150–200 g. Two rats per cage were housed in a temperature controlled room at 24 6 1 C on a 12:12 h dark/light cycle, with free access to water and food. Food intake and animal growth were monitored daily. They were allowed to have unrestricted movement in their housing. Rats were randomly divided into three groups of eight rats each.
Group 1 (Pinealectomy Group) A pinealectomy was performed and the sciatic nerve dissected and coapted during the same session. After nerve coaptation, 0.5 mL 0.9% NaCl (Eczacıbasi-Baxter Drug Industry, Istanbul, Turkey) was injected intraperitoneally. We repeated this last procedure between 10 and 11 AM, daily, for the 21 d.
Group 2 (Melatonin Group) We performed a craniotomy without pinealectomy. The sciatic nerve was dissected and repaired during the same session. After nerve coaptation, 0.5 mL melatonin (Interlab A.S., Sigma-Aldrich Local Office, Istanbul, Turkey) was injected intraperitoneally (10 mg/kg 1% 0.5 mL). We repeated this last procedure, between 10 and 11 AM, daily, for 21 d.
Group 3 (Control Group) A craniotomy was performed without pinealectomy. The sciatic nerve was dissected and repaired during the same session. After nerve coaptation, 0.5 mL 0.9% NaCl was injected intraperitoneally. We repeated this last procedure, between 10 and 11 AM, daily, for 21 d. In each group, nerve recovery was evaluated histologically, functionally, and electrophysiologically. Functional and electrophysiologic evaluations were conducted before the initial surgery and at 4 and 12 wk.
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Evaluation Histologic Evaluation At 12 wk, after completing electrophysiological and functional evaluation, the sciatic nerve was dissected, including the nerve repair line, with surrounding tissue from the ischiadic notch to the popliteal fossa. After this, rats were sacrificed by injecting high-dose intracardiac thiopental sodium (pentonal sodium, ampule 0.5 g; Abbott Laboratory, Istanbul, Turkey). Removed tissue was fixed with 10% formalin, dehydrated with alcohol solution, and embedded in paraffin. Transverse sections were taken 0.5 cm distal and proximal to the repair line. Longitudinal sections were taken in 5 mm thicknesses from the repair line. The sections were stained with hematoxylin and eosin (H and E) and Masson’s trichrome for evaluation of collagen, as well.
Functional Evaluation Walking track analysis. Walking track analysis was performed in all rats at 4 and 12 wk, or until the rats were sacrificed. As defined in other studies, rats were allowed to walk in a dark, single-ended, closed corridor, which was 10 3 10 3 100 cm in size. Absorbent paper was placed on the bottom of this corridor. Prior to being permitted to walk, the legs of these rats were dipped in methylene blue. Measurements were made by following the rat’s footprints (Fig. 1). Walking track analysis was calculated as follows: (1) Print length factor (PLF): [experimental print length (EPL)normal print length (NPL)] /NPL (2) Toe spread factor (TSF): [experimental toe spread (ETS)normal toe spread (NTS)]/NTS (3) Intermediary toe spread factor (ITF): [experimental intermediary toe spread (EIT)-normal intermediary toe spread (NIT)/NIT] These three factors then were used to calculate the Bain Mackinnon Hunter (BMH) sciatic function index (SFI), using the formula: SFI ¼ 38.3 (PLF) þ 109.5 (TSF) þ 13.3 (ITF) 8.8, where an SFI of 0 represents normal function, and an SFI of –100 demonstrates complete functional loss.
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FIG. 1. Walking track analysis of the animals. Typical walking track analysis pattern obtained from a rat with sciatic nerve anastomosis. Figure shows a normal sciatic nerve analysis in the right control leg (B) and a repaired sciatic nerve with anastomosis in the left leg (A). NTS ¼ normal toe spread; NIT ¼ normal intermediary toe spread; NPL ¼ normal print length; ETS ¼ experimental toe spread, EIT ¼ experimental intermediary toe spread; EPL ¼ experimental print length.
Electrophysiologic Evaluation In each rat, compound muscle action potentials (CMAP) were measured. Electrophysiological records were performed using a Medtronic Keypoint version 3.0 device and Keypoint Software version 3.00 (Medtronic International Trading, Tolochenaz, Switzerland). For sciatic nerve stimulation, bipolar tungsten metal electrodes were used, with the cathodes positioned distally. Recording was conducted using superficial electrodes, which were 0.5 cm in size, and composed of Ag/ AgCL, with the active electrode on the gastrocnemius muscle and the reference electrode on the tendon of the same muscle. The ground electrode was placed on the shaved tail. The highest response with lowest amplitude was recorded. The duration of the stimulus was 0.1 ms, with the band range of the filter set between 3 and 10 Hz. Latency, amplitude, and duration of the CMAP were measured.
Statistics Between-group differences at baseline and at 4 and 12 wk were evaluated by means of Mann-Whitney U tests. Data were presented as means 6 standard deviations (SD). All data were analyzed using SPSS for Windows version 14.0 (SPSS Inc., Chicago, IL) with differences associated with P < 0.05 interpreted as statistically significant.
RESULTS Histopathologic Findings
Along the repair line, in the sections that were stained with Masson’s trichome, within the pinealectomy group, the repair area was badly organized and the diameter of the extrafascicular connective tissue increased relative to the other two groups. This extrafascicular connective tissue (epineurium) extended inside the tissue. The amount of endoneural collagen was markedly increased in this group (Fig. 2A). In the control group, the endoneural collagen and epineurium diameter were increased, but this was less than that of in
the pinealectomy group (Fig. 2C). In the melatonin group, the epineurium was thinner, there was less endoneural collagen, and the collagen was better organized (Fig. 2E). Examining the sections distal to the suture line that were stained with H and E, in the pinealectomy group, the number of axons was markedly reduced and hyperchromatically-stained demyelinated axons were observed (Fig. 2B). In the control group, we observed similar findings (Fig. 2D). However, in the melatonin group, there was less of a decrease in the number of axons, and there were fewer demyelinized axons. In addition, the nerve fibers exhibited better organization (Fig. 2F). Walking Track Analysis
Sciatic functional indices (SFI) at 4 and 12 wk are shown in Table 1. At 4 wk, there were no significant differences observed in any of the groups versus baseline; however, by the 12 wk, a prominent and statistically significant difference was observed in the melatonin group relative to the other two groups. The SFI scores were similar in the pinealectomy and control groups (Tables 1 and 2). Electrophysiologic Findings
In all rats, action potential amplitude, latency, and nerve conduction velocity were recorded just before dissecting the sciatic nerves and at 12 wk; the results are shown in Table 3. Prior to cutting the sciatic nerve, there were no inter-group differences in these parameters. However, at 12 wk, in each group, latencies were prolonged, and amplitudes and nerve conduction velocities decreased. Inter-group comparisons are shown in Table 4. Significant differences were seen in the melatonin group with respect to each parameter, compared with the other groups. However, there were no significant differences between the pinealectomy and control groups in any of the three parameters (Tables 3 and 4). DISCUSSION
Degeneration occurs when injuries to the peripheral nerves disrupt axonal continuity. Wallerian degeneration occurs from the site of injury to the distal end of the nerve, and defines the destruction of the axon and surrounding myelin. Just proximal to the injury, there is a limited degeneration. Even if a dissected or injured nerve is surgically repaired after an injury, there is another inevitable problem, which is scar tissue formation. Scar tissue can physically impede the forward growth of the axon, causing branching, disconnection, backtracking, or even termination of the growth of the
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FIG. 2. Histologic photomicrographs of sagittal and axial sections of the sciatic nerve in groups. Sagittal section of the collagen formation areas in the anastomosis region of the sciatic nerve in pinealectomy (A), control (C), and MLT-treated group (E). Note that the amount of endoneural collagen (white arrows) was markedly increased in pinealectomy and control groups compared with the MLT-treated group (Masson’s trichrome 3200). Axial sections distal to the suture line in the groups show axonal regeneration sprouts. Note that the number of axons was markedly reduced and hyperchromatically stained demyelinated axons in the pinealectomy (B) and control (D) group compared with the MLTtreated (F) group (H and E 3400).
nerve growth cone [1, 5, 6, 15]. Many factors play a role in nerve repair after an injury, including the surgical technique used, the duration of time between the injury TABLE 1 The SFI Scores in the Pinealectomy, Melatonin, and Control Groups 4th wk
12th wk
and surgical repair, several growth factors and mediators, the impossibility of joining all axons to distal ones, and scar tissue formation [3, 5, 16–18]. Therefore, after a major nerve injury, even after successful nerve repair, complete functional restoration is impossible. Melatonin has a stronger free radical removal effect than all other known antioxidants (mannitol, glutathione, and vitamin E) [11, 13]. Moreover, contrary to
Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 (P) (M) (C) (P) (M) (C) 1 86.4 2 82.3 3 84.7 4 87.5 5 87.7 6 92.2 7 86.3 8 85.5 Mean 6 86.58 6 SD 2.84
82.4 84.9 85.6 80.6 77.3 84.8 83.2 87.2 83.25 6 3.15
83.8 86.8 87.3 85.5 86.7 84.5 85.9 87.1 85.95 6 1.27
69.3 66.4 67.3 69.8 68.5 71.3 68.2 64.1 68.11 6 2.21
60.2 60.2 61.8 58.2 53.4 61.9 60.7 62.2 59.83 6 2.90
68.8 67.9 69.5 64.8 65.4 68.4 73.3 68.2 68.28 6 2.61
TABLE 2 Statistical Results of the SFI Scores in Inter-Group Comparisons (Mann-Whitney U test) Group 1(P)- 2(M)-3(C) (n ¼ 24)
P values before sciatic nerve cut/injury
P values at 12th wk
P-M P-C M-C
P ¼ 0.059 P ¼ 0.636 P ¼ 0.059
P ¼ 0.001 P ¼ 0.958 P ¼ 0.001
P ¼ pinealectomy group; M ¼ melatonin group, C ¼ control group.
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TABLE 3 Electrophysiological Findings in all Groups Before nerve injury
Latency (ms)
1 2 3 4 5 6 7 8 Amplitude (mV) 1 2 3 4 5 6 7 8 Nerve conduction 1 velocity (m/mc) 2 3 4 5 6 7 8
At 12th week
P
M
C
P
M
C
1.08 1.08 1.08 .92 1.33 .75 1.17 .83 67.10 36.00 45.20 52.50 54.10 36.50 16.70 30.90 40.00 40.00 40.00 62.50 58.80 58.80 62.50 40.00
.83 1.00 .83 .83 .83 .83 1.08 .92 41.50 41.70 67.00 42.90 55.70 64.60 54.50 14.20 58.80 62.50 58.80 58.80 58.80 58.80 58.80 62.50
.75 1.08 1.25 .83 1.08 .92 1.00 1.25 42.70 27.90 34.30 67.40 45.80 60.80 20.10 57.30 40.00 58.80 58.80 58.80 58.80 62.50 58.80 58.80
1.67 1.25 1.42 1.08 1.58 1.08 1.33 1.08 13.40 26.20 21.40 21.10 17.70 27.90 13.40 20.10 4.00 23.80 12.80 20.00 30.30 29.40 50.70 20.00
.83 1.00 1.00 1.33 1.00 .83 1.33 1.08 39.50 31.80 54.20 36.50 39.50 45.80 37.70 13.40 40.00 58.80 58.80 40.00 40.00 40.00 40.00 58.80
1.58 1.25 1.42 1.17 2.00 1.00 1.92 1.25 20.30 26.20 23.70 30.00 18.00 35.80 8.90 1.60 23.80 40.00 40.00 24.40 33.30 40.00 35.70 23.80
P ¼ pinealectomy group; M ¼ melatonin group; C ¼ control group.
other antioxidants, melatonin dissolves both in water and in lipids; therefore, it reaches all cell components, where it can act as an antioxidant. In brain ischemia and reperfusion models, melatonin has been shown to decrease damage [10–13, 19, 20]. These changes were attributed to the antioxidative capacity of melatonin. In human studies, melatonin has been given in a very large range of doses, from 0.1 mg to 2000 mg. The large doses are consistent with animal studies, demonstrating the very low toxicity of this hormone, even at pharmacologic doses; doses over 0.5 mg are accepted as TABLE 4 Statistical Results of the Electrophysiological Study in Group Comparisons (Mann-Whitney U test) Grup 1(P)-2(M) -3(C) (n ¼ 24) Latency (ms)
Amplitude (mV)
Nerve conduction velocity (m/ms)
P-M P-C M-C P-M P-C M-C P-M P-C M-C
P values before nerve repairing
P values at 12th week
P ¼ 0.110 P ¼ 0.873 P ¼ 0.143 P ¼ 0.400 P ¼ 0.753 P ¼ 0.834 P ¼ 0.170 P ¼ 0.423 P ¼ 0.332
P ¼ 0.022 P ¼ 0.526 P ¼ 0.023 P ¼ 0.008 P ¼ 0.713 P ¼ 0.009 P ¼ 0.004 P ¼ 0.072 P ¼ 0.006
P ¼ pinealectomy group; M ¼ melatonin group; C ¼ control group.
pharmacologic [8, 21]. Melatonin displays its antioxidant effects by means of two mechanisms. The first is through its direct influence on toxic radicals, and this is independent of receptors. This feature depends upon its strong binding of hydroxyl and peroxyl radicals. The second effect is through the receptors over the genomes. Through these receptors, it induces enzymes that detoxify free radicals [10–22]. However, both the direct and indirect effects do not occur sufficiently at physiologic levels. It has been suggested that the antioxidant effects can be seen more during the significantly higher concentrations associated with nocturnal peak serum concentrations [8]. Melatonin has many other known effects that pertain to our study hypothesis other than its antioxidant effect [10, 11]. In experimental models, intracellular calcium levels have a central role in nerve recovery. Venacek et al. and Angelov et al. have shown that melatonin prevents intracellular calcium influx by influencing voltage-dependent calcium channels, thereby mimicking the effects of Ca channel blockers [23, 24]. Based upon the above-mentioned information, we know that even if rapid and successful repair nerve is achieved, complete functional restoration is not feasible. Too many inevitable factors prevent complete recovery. One of these is disruption of intraneural microcirculation and the subsequent development of ischemia along the repair line. During ischemia, the concentration of calcium ions increases in the cytosol, cell swelling develops, and toxic free radicals increase in number, all of which lead to neural tissue damage. We propose that melatonin exerts its nerve protection effect by preventing intracellular calcium influx and free radical-induced damage. In this study, we evaluated the effects of melatonin on nerve healing in several ways: functionally, electrophysiologically, and histologically. Histologic evaluation revealed prominent improvement in the melatonin group, demonstrating melatonin’s positive effect on nerve tissue repair. However, even though there was marked improvement in the control group, there was no significant improvement relative to the pinealectomy group. Weichselbaum et al. demonstrated that in pinealectomized rats, wound healing is slower than in controls, and in rats given melatonin, faster [25]. Drobnik et al. found that pinealectomy increased the amount of collagen, whereas melatonin, given in doses of 30–100 ug, decreased the collagen amount [26]. These findings are consistent with our findings. In our study, the amount of collagen laid down was greatest in the pinealectomy group and its organization was worst. In controls, a nonsignificant decrease in collagen amount was noted; but rats that received melatonin had significantly less and better-organized collagen. Even with a technically excellent nerve repair,
ATIK ET AL.: MELATONIN AND NERVE REGENERATION
scar formation is inevitable. Inhibition of collagen formation contributes meaningfully to nerve recovery. These findings show us that melatonin has little effect in physiologic doses. The desired effect of melatonin is achieved only when it is given in high enough doses, whether that by local or systemic administration. The second segment of our study involved a functional evaluation, which consisted of a walking track analysis. Selection of appropriate evaluation methods is crucial when measuring experimental nerve recovery. In 1982, De Medinaceli first reported that the SFI could be used to evaluate total lower limb function, including nerve, muscle, and joint function in rats [27]. Since then, many investigators have used walking track analysis as an assessment of global function recovery after sciatic nerve injuries or repairs. The use of walking track analysis provides a noninvasive method by which to assess the functional status of the sciatic nerve, avoiding interaction with the regeneration process, based on measurement of footprints, expressed in units of functional deficit. Because proper walking requires coordinated functions, including sensory input, motor responses, and cortical integration, SFI may be better than extrapolating from the basic electrophysiology and histomorphometry of axon growth and muscle innervation, especially if the research focus concerns functional outcome [28]. In our study, the results of walking track analysis supported the histologic findings. This analysis revealed significant improvement in the melatonin group, but no significant difference between controls and rats that had undergone a pinealectomy, again showing us that a desirable effect can only be achieved with melatonin doses producing higher than physiologic levels. The third and final part of our study was an electrophysiologic evaluation. In this evaluation, the only inter-group difference and difference versus baseline occurred in the melatonin group at 12 wk data collection point, suggesting again that melatonin is effective only when given in supraphysiologic doses; moreover, it needs to be given over a relatively long period. Combining these findings together, we conclude that melatonin has a significant beneficial effect on nerve recovery, that this effect is apparent functionally, histopathologically and electrophysiologically, and that the results observed in each of these three areas appear to be consistent. However, for these effects to occur, the dose of melatonin must be high enough to achieve supraphysiologic levels of the hormone.
CONCLUSIONS
Peripheral nerve injuries continue to be a major clinical challenge, so that the search for better treatments
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and a better understanding of nerve injury and repair is ongoing. Several surgical techniques and medical agents have been tried, in an attempt to establish some consensus as to the most effective treatment protocol. Our study provides a novel insight into this unclear field, in that it illustrates melatonin’s potential as a nerve reparative agent. The beneficial effects of melatonin cannot be achieved at physiologic doses, however. Higher doses and a longer duration of administration are necessary. Future research is warranted to compare melatonin’s effects versus those of other nerve-regenerating agents, and ultimately to explore the effectiveness of melatonin in human nerve injury patients within the context of a randomized, doubleblinded clinical trial. REFERENCES 1. Brushart TM. Nerve repair and grafting. In: Green DP, Ed. Operative hand surgery. 4th ed. Edinburg: Churchill Livingstone, 1999. p. 1381. 2. Terzis JK, Smith KL. Repair and grafting of the peripheral nerve. In: McCarthy JG, Ed. Plastic surgery. Philadelphia: WB Saunders, 1990. p. 630. 3. Sunderland S. The history of nerve repair. A critical appraisal. Edinburgh: Churchill Livingstone, 1991. 4. Lundborg G. Nerve regeneration and repair. A review. Acta Orthop Scand 1987;58:145. 5. Sunderland S. Nerves and nerve injuries. Edinburgh: Churchill Livingstone, 1978. 6. Myers RR, Yamamoto T, Yaksh TL, et al. The role of focal nerve ischemia and Wallerian degeneration in peripheral nerve injury producing hyperesthesia. Anesthesiology 1993;78:308. 7. Kater SB, Mills LR. Regulation of growth cone behavior by calcium. J Neurosci 1991;11:891. 8. Brzezinski A. Melatonin in humans. N Engl J Med 1997;336:186. 9. Cagnacci A. Melatonin in relation to physiology in adult humans. J Pineal Res 1996;21:200. 10. Pierrefiche G, Zerbib R, Laborit H. Anxiolytic activity of melatonin in mice: Involvement of benzodiazepine receptors. Res Commun Chem Pathol Pharmacol 1993;82:131. 11. Antolin I, Rodriguez C, Sainz RM, et al. Neurohormone melatonin prevents cell damage: Effect on gene expression for antioxidant enzymes. FASEB J 1996;10:882. 12. Pieri C, Marra M, Moroni F, et al. Melatonin: A peroxyl radical scavenger more effective than vitamin E. Life Sci 1994; 55:PL271. 13. Poeggeler B, Saarela S, Reiter RJ, et al. Melatonin—A highly potent endogenous radical scavenger and electron donor: New aspects of the oxidation chemistry of this indole accessed in vitro. Ann N Y Acad Sci 1994;738:419. 14. Pohlymeyer G, Reuss S, Baum A. An improved technique for visually controlled pinealectomy in the rat. J Exp Anim Sci 1994; 36:84. 15. Komiyama A, Novicki DL, Suzuki K. Adhesion and proliferation are enhanced in vitro in Schwann cells from nerve undergoing Wallerian degeneration. J Neurosci Res 1991;29:308. 16. Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res 1982;232:157. 17. Van der Zee CE, Brakkee JH, Gispen WH. Putative neurotrophic factors and functional recovery from peripheral nerve damage in the rat. Br J Pharmacol 1991;103:1041. 18. Lipton SA. Growth factors for neuronal survival and process regeneration. Implications in the mammalian central nervous system. Arch Neurol 1989;46:1241.
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19. Kotler M, Rodriguez C, Sainz RM, et al. Melatonin increases gene expression for antioxidant enzymes in rat brain cortex. J Pineal Res 1998;24:83. 20. Kilic E, Ozdemir YG, Bolay H, et al. Pinealectomy aggravates and melatonin administration attenuates brain damage in focal ischemia. J Cereb Blood Flow Metab 1999;19:511. 21. Sack RL, Lewy AJ, Hughes RJ. Use of melatonin for sleep and circadian rhythm disorders. Ann Med 1998;30:115. 22. Princ FG, Maxit AG, Cardalda C, et al. In vivo protection by melatonin against delta-aminolevulinic acid-induced oxidative damage and its antioxidant effect on the activity of haem enzymes. J Pineal Res 1998;24:1. 23. Vanecek J. Cellular mechanisms of melatonin action. Physiol Rev 1998;78:687.
24. Angelov DN, Neiss WF, Streppel M, et al. Nimodipine accelerates axonal sprouting after surgical repair of rat facial nerve. J Neurosci 1996;16:1041. 25. Weichselbaum R, Patel M, Das Gupta TK. Influence of the pineal on wound healing. Nature 1975;254:349. 26. Drobnik J, Dabrowski R. Melatonin suppresses the pinealectomy-induced elevation of collagen content in a wound. Cytobios 1996;85:51. 27. de Medinaceli L, Freed WJ, Wyatt RJ. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp Neurol 1982;77:634. 28. Varejao AS, Meek MF, Ferreira AJ, et al. Functional evaluation of peripheral nerve regeneration in the rat: Walking track analysis. J Neurosci Methods 2001;108:1.
The Effects of Exogenous Melatonin on Peripheral Nerve Regeneration and Collagen Formation in Rats Bekir Atik, M.D.,* Ibrahim Erkutlu, M.D.,† Mustafa Tercan, M.D.,k Hakan Buyukhatipoglu, M.D.,{,1 Mehmet Bekerecioglu, M.D.,‡ and Sadrettin Pence, M.D., Ph.D.§ *Department of Plastic and Reconstructive Surgery, Yuzuncu Yil University, Medical Faculty, Van, Turkey; †Department of Neurosurgery; ‡Department of Plastic and Reconstructive Surgery; §Department of Physiology, Medical Faculty, Gaziantep University, Gaziantep, Turkey; kDepartment of Plastic and Reconstructive Surgery, Haydarpasa Numune Hospital, Istanbul, Turkey; and {Department of Internal Medicine, Harran University School of Medicine, Sanliurfa, Turkey Submitted for publication March 16, 2009
Background. Peripheral nerve damage that requires surgical repair does not result in complete recovery because of collagen scar formation, ischemia, free oxygen radical damage, and other factors. To date, the best treatment method has not yet been determined. In this study, we designed an experimental peripheral nerve injury model, and researched the possible effects of melatonin hormone, based on evidence of its strong antioxidant and cell-protective effects via mimicking the effects of calcium channel blockers. Materials and Methods. We randomized 24 healthy female albino rats into three groups: the pinealectomy group, melatonin group, and control group. In the pinealectomy group, craniotomy, pinealectomy, sciatic nerve transection, and coaptation were performed, and 0.9% NaCl was injected intraperitoneally. In the melatonin group, craniotomy (without pinealectomy), sciatic nerve dissection, and coaptation were performed, and melatonin was injected intraperitoneally, instead of NaCl. In the control group, craniotomy (without pinealectomy), sciatic nerve dissection and coaptation, and intraperitoneal NaCl injection were performed. In each group, nerve recovery was evaluated histologically, functionally, and electrophysiologically. Functional and electrophysiologic evaluations were conducted before surgery and at 4 and 12 wk. Results. At 4 wk, no significant difference was observed between the groups. However, at 12 wk, significant electrophysiologic and functional improvement was observed only in the melatonin group.
1 To whom correspondence and reprint requests should be addressed at Harran University School of Medicine, Gazimuhtarpasa bul. Kalyon is merkezi No.17-18 Sehitkamil, Gaziantep, Turkey. E-mail: [email protected], [email protected].
0022-4804/$36.00 Ó 2011 Elsevier Inc. All rights reserved.
Conclusions. Melatonin seems to have a beneficial effect on nerve recovery. However, this effect is not effective at physiologic doses. Future comparative studies with melatonin versus other nerve-regenerating agents are necessary to determine the clinical utility of melatonin hormone. Ó 2011 Elsevier Inc. All rights reserved. Key Words: peripheral nerve injury; regeneration; melatonin; collagen formation.
INTRODUCTION
Peripheral nerves are the most important structures for providing motor and sensory function in mammalians. Damage to peripheral nerves results in partial or complete loss of these functions. Currently, peripheral nerve injuries and repairs continue to be a major problem. Surgical reconstruction procedures do not yield complete recovery. Many studies have been performed on surgical techniques; however, studies on medications that might positively affect peripheral nerve regeneration are rare [1–4]. When damage occurs to a peripheral nerve, both ischemic and inflammatory processes begin. As part of this process, free oxygen radicals and many toxic agents accumulate around the site of injury. As a result, membrane permeability is disturbed and intracellular calcium influx is stimulated. Intracellular calcium influx, in turn, activates proteolytic pathways, resulting in cell destruction, which include the destruction of neurofilaments and microtubules. Another important point relevant to the regenerative process is that scar tissue forms around the regeneration field. Any scar tissue
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that forms between the proximal and distal ends of a repaired nerve can physically impede the forward development of the axon [3, 5–7]. In this study, we researched the role of the hormone melatonin in the regenerative process. Melatonin is secreted by the pineal gland that lies at the base of the brain. Considerable evidence exists to document the many different roles of melatonin in the human body, including its importance in circadian rhythms, sleep physiology, mental status, reproduction, tumor development, aging, and many other physiologic processes. Its many functions in the human body have not yet been completely understood [8–10]. Many in vivo and in vitro studies have demonstrated that melatonin is a strong free radical removal agent. Melatonin can protect macromolecules, especially DNA, from oxidative damage. It exerts this effect directly, independent of receptors [11–13]. In order to research melatonin’s role in the nerve regeneration process, we designed an experimental peripheral nerve injury model.
Surgical Techniques Rats were starved for 24 h prior to surgery. Anesthesia was induced using xylocaine (Rhompun, 2%; Bayer Drug Industry, Istanbul, Turkey) 10 mg/kg and ketamine hydrochloride (Katalar, 5%; Eczacıbas xı Drug Industry with permission from Parke-Davis, Ann Arbor, MI), which were mixed and given intraperitoneally. Microsurgical techniques were utilized under sterile conditions, using 4 3 4 magnification surgery loops.
Pinealectomy Anesthetized rats were affixed to the operation table at each of their four extremities, as described by Pohlymeyer et al. [14]. Heads and necks were cleaned gently with polyvinylpyrolidon iodine (polyod 10% solution; Drogsan Drug Industry, Ankara, Turkey) and covered with a sterile surgical drape. A 1.5 cm incision was made over the hairless scalp, just behind the eyes. Through this incision, the pineal gland was carefully removed.
Sciatic Nerve Coaptation With the rat in the same position, the sacrum and left lower extremities were shaved and cleaned with polyvinylpyrolidon. Under surgical conditions, a skin incision was made parallel to the long extremity axis. The gluteus maximus muscle was dissected from the femoral bone edges, and the sciatic nerve exposed. The nerve was freed from surrounding tissues. The tibial and peroneal branches were bluntly dissected from each other. Next, the nerve was dissected 15 mm cephalad to the sural, peroneal, and tibial bifurcations. Dissected nerve ends were coapted with prolene suture material.
MATERIAL AND METHODS Study Design and Groups This study was approved by the local ethics committee of Gaziantep University School of Medicine. In this study, we used 24 healthy female albino rats, each weighing 150–200 g. Two rats per cage were housed in a temperature controlled room at 24 6 1 C on a 12:12 h dark/light cycle, with free access to water and food. Food intake and animal growth were monitored daily. They were allowed to have unrestricted movement in their housing. Rats were randomly divided into three groups of eight rats each.
Group 1 (Pinealectomy Group) A pinealectomy was performed and the sciatic nerve dissected and coapted during the same session. After nerve coaptation, 0.5 mL 0.9% NaCl (Eczacıbasi-Baxter Drug Industry, Istanbul, Turkey) was injected intraperitoneally. We repeated this last procedure between 10 and 11 AM, daily, for the 21 d.
Group 2 (Melatonin Group) We performed a craniotomy without pinealectomy. The sciatic nerve was dissected and repaired during the same session. After nerve coaptation, 0.5 mL melatonin (Interlab A.S., Sigma-Aldrich Local Office, Istanbul, Turkey) was injected intraperitoneally (10 mg/kg 1% 0.5 mL). We repeated this last procedure, between 10 and 11 AM, daily, for 21 d.
Group 3 (Control Group) A craniotomy was performed without pinealectomy. The sciatic nerve was dissected and repaired during the same session. After nerve coaptation, 0.5 mL 0.9% NaCl was injected intraperitoneally. We repeated this last procedure, between 10 and 11 AM, daily, for 21 d. In each group, nerve recovery was evaluated histologically, functionally, and electrophysiologically. Functional and electrophysiologic evaluations were conducted before the initial surgery and at 4 and 12 wk.
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Evaluation Histologic Evaluation At 12 wk, after completing electrophysiological and functional evaluation, the sciatic nerve was dissected, including the nerve repair line, with surrounding tissue from the ischiadic notch to the popliteal fossa. After this, rats were sacrificed by injecting high-dose intracardiac thiopental sodium (pentonal sodium, ampule 0.5 g; Abbott Laboratory, Istanbul, Turkey). Removed tissue was fixed with 10% formalin, dehydrated with alcohol solution, and embedded in paraffin. Transverse sections were taken 0.5 cm distal and proximal to the repair line. Longitudinal sections were taken in 5 mm thicknesses from the repair line. The sections were stained with hematoxylin and eosin (H and E) and Masson’s trichrome for evaluation of collagen, as well.
Functional Evaluation Walking track analysis. Walking track analysis was performed in all rats at 4 and 12 wk, or until the rats were sacrificed. As defined in other studies, rats were allowed to walk in a dark, single-ended, closed corridor, which was 10 3 10 3 100 cm in size. Absorbent paper was placed on the bottom of this corridor. Prior to being permitted to walk, the legs of these rats were dipped in methylene blue. Measurements were made by following the rat’s footprints (Fig. 1). Walking track analysis was calculated as follows: (1) Print length factor (PLF): [experimental print length (EPL)normal print length (NPL)] /NPL (2) Toe spread factor (TSF): [experimental toe spread (ETS)normal toe spread (NTS)]/NTS (3) Intermediary toe spread factor (ITF): [experimental intermediary toe spread (EIT)-normal intermediary toe spread (NIT)/NIT] These three factors then were used to calculate the Bain Mackinnon Hunter (BMH) sciatic function index (SFI), using the formula: SFI ¼ 38.3 (PLF) þ 109.5 (TSF) þ 13.3 (ITF) 8.8, where an SFI of 0 represents normal function, and an SFI of –100 demonstrates complete functional loss.
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FIG. 1. Walking track analysis of the animals. Typical walking track analysis pattern obtained from a rat with sciatic nerve anastomosis. Figure shows a normal sciatic nerve analysis in the right control leg (B) and a repaired sciatic nerve with anastomosis in the left leg (A). NTS ¼ normal toe spread; NIT ¼ normal intermediary toe spread; NPL ¼ normal print length; ETS ¼ experimental toe spread, EIT ¼ experimental intermediary toe spread; EPL ¼ experimental print length.
Electrophysiologic Evaluation In each rat, compound muscle action potentials (CMAP) were measured. Electrophysiological records were performed using a Medtronic Keypoint version 3.0 device and Keypoint Software version 3.00 (Medtronic International Trading, Tolochenaz, Switzerland). For sciatic nerve stimulation, bipolar tungsten metal electrodes were used, with the cathodes positioned distally. Recording was conducted using superficial electrodes, which were 0.5 cm in size, and composed of Ag/ AgCL, with the active electrode on the gastrocnemius muscle and the reference electrode on the tendon of the same muscle. The ground electrode was placed on the shaved tail. The highest response with lowest amplitude was recorded. The duration of the stimulus was 0.1 ms, with the band range of the filter set between 3 and 10 Hz. Latency, amplitude, and duration of the CMAP were measured.
Statistics Between-group differences at baseline and at 4 and 12 wk were evaluated by means of Mann-Whitney U tests. Data were presented as means 6 standard deviations (SD). All data were analyzed using SPSS for Windows version 14.0 (SPSS Inc., Chicago, IL) with differences associated with P < 0.05 interpreted as statistically significant.
RESULTS Histopathologic Findings
Along the repair line, in the sections that were stained with Masson’s trichome, within the pinealectomy group, the repair area was badly organized and the diameter of the extrafascicular connective tissue increased relative to the other two groups. This extrafascicular connective tissue (epineurium) extended inside the tissue. The amount of endoneural collagen was markedly increased in this group (Fig. 2A). In the control group, the endoneural collagen and epineurium diameter were increased, but this was less than that of in
the pinealectomy group (Fig. 2C). In the melatonin group, the epineurium was thinner, there was less endoneural collagen, and the collagen was better organized (Fig. 2E). Examining the sections distal to the suture line that were stained with H and E, in the pinealectomy group, the number of axons was markedly reduced and hyperchromatically-stained demyelinated axons were observed (Fig. 2B). In the control group, we observed similar findings (Fig. 2D). However, in the melatonin group, there was less of a decrease in the number of axons, and there were fewer demyelinized axons. In addition, the nerve fibers exhibited better organization (Fig. 2F). Walking Track Analysis
Sciatic functional indices (SFI) at 4 and 12 wk are shown in Table 1. At 4 wk, there were no significant differences observed in any of the groups versus baseline; however, by the 12 wk, a prominent and statistically significant difference was observed in the melatonin group relative to the other two groups. The SFI scores were similar in the pinealectomy and control groups (Tables 1 and 2). Electrophysiologic Findings
In all rats, action potential amplitude, latency, and nerve conduction velocity were recorded just before dissecting the sciatic nerves and at 12 wk; the results are shown in Table 3. Prior to cutting the sciatic nerve, there were no inter-group differences in these parameters. However, at 12 wk, in each group, latencies were prolonged, and amplitudes and nerve conduction velocities decreased. Inter-group comparisons are shown in Table 4. Significant differences were seen in the melatonin group with respect to each parameter, compared with the other groups. However, there were no significant differences between the pinealectomy and control groups in any of the three parameters (Tables 3 and 4). DISCUSSION
Degeneration occurs when injuries to the peripheral nerves disrupt axonal continuity. Wallerian degeneration occurs from the site of injury to the distal end of the nerve, and defines the destruction of the axon and surrounding myelin. Just proximal to the injury, there is a limited degeneration. Even if a dissected or injured nerve is surgically repaired after an injury, there is another inevitable problem, which is scar tissue formation. Scar tissue can physically impede the forward growth of the axon, causing branching, disconnection, backtracking, or even termination of the growth of the
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FIG. 2. Histologic photomicrographs of sagittal and axial sections of the sciatic nerve in groups. Sagittal section of the collagen formation areas in the anastomosis region of the sciatic nerve in pinealectomy (A), control (C), and MLT-treated group (E). Note that the amount of endoneural collagen (white arrows) was markedly increased in pinealectomy and control groups compared with the MLT-treated group (Masson’s trichrome 3200). Axial sections distal to the suture line in the groups show axonal regeneration sprouts. Note that the number of axons was markedly reduced and hyperchromatically stained demyelinated axons in the pinealectomy (B) and control (D) group compared with the MLTtreated (F) group (H and E 3400).
nerve growth cone [1, 5, 6, 15]. Many factors play a role in nerve repair after an injury, including the surgical technique used, the duration of time between the injury TABLE 1 The SFI Scores in the Pinealectomy, Melatonin, and Control Groups 4th wk
12th wk
and surgical repair, several growth factors and mediators, the impossibility of joining all axons to distal ones, and scar tissue formation [3, 5, 16–18]. Therefore, after a major nerve injury, even after successful nerve repair, complete functional restoration is impossible. Melatonin has a stronger free radical removal effect than all other known antioxidants (mannitol, glutathione, and vitamin E) [11, 13]. Moreover, contrary to
Group 1 Group 2 Group 3 Group 1 Group 2 Group 3 (P) (M) (C) (P) (M) (C) 1 86.4 2 82.3 3 84.7 4 87.5 5 87.7 6 92.2 7 86.3 8 85.5 Mean 6 86.58 6 SD 2.84
82.4 84.9 85.6 80.6 77.3 84.8 83.2 87.2 83.25 6 3.15
83.8 86.8 87.3 85.5 86.7 84.5 85.9 87.1 85.95 6 1.27
69.3 66.4 67.3 69.8 68.5 71.3 68.2 64.1 68.11 6 2.21
60.2 60.2 61.8 58.2 53.4 61.9 60.7 62.2 59.83 6 2.90
68.8 67.9 69.5 64.8 65.4 68.4 73.3 68.2 68.28 6 2.61
TABLE 2 Statistical Results of the SFI Scores in Inter-Group Comparisons (Mann-Whitney U test) Group 1(P)- 2(M)-3(C) (n ¼ 24)
P values before sciatic nerve cut/injury
P values at 12th wk
P-M P-C M-C
P ¼ 0.059 P ¼ 0.636 P ¼ 0.059
P ¼ 0.001 P ¼ 0.958 P ¼ 0.001
P ¼ pinealectomy group; M ¼ melatonin group, C ¼ control group.
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TABLE 3 Electrophysiological Findings in all Groups Before nerve injury
Latency (ms)
1 2 3 4 5 6 7 8 Amplitude (mV) 1 2 3 4 5 6 7 8 Nerve conduction 1 velocity (m/mc) 2 3 4 5 6 7 8
At 12th week
P
M
C
P
M
C
1.08 1.08 1.08 .92 1.33 .75 1.17 .83 67.10 36.00 45.20 52.50 54.10 36.50 16.70 30.90 40.00 40.00 40.00 62.50 58.80 58.80 62.50 40.00
.83 1.00 .83 .83 .83 .83 1.08 .92 41.50 41.70 67.00 42.90 55.70 64.60 54.50 14.20 58.80 62.50 58.80 58.80 58.80 58.80 58.80 62.50
.75 1.08 1.25 .83 1.08 .92 1.00 1.25 42.70 27.90 34.30 67.40 45.80 60.80 20.10 57.30 40.00 58.80 58.80 58.80 58.80 62.50 58.80 58.80
1.67 1.25 1.42 1.08 1.58 1.08 1.33 1.08 13.40 26.20 21.40 21.10 17.70 27.90 13.40 20.10 4.00 23.80 12.80 20.00 30.30 29.40 50.70 20.00
.83 1.00 1.00 1.33 1.00 .83 1.33 1.08 39.50 31.80 54.20 36.50 39.50 45.80 37.70 13.40 40.00 58.80 58.80 40.00 40.00 40.00 40.00 58.80
1.58 1.25 1.42 1.17 2.00 1.00 1.92 1.25 20.30 26.20 23.70 30.00 18.00 35.80 8.90 1.60 23.80 40.00 40.00 24.40 33.30 40.00 35.70 23.80
P ¼ pinealectomy group; M ¼ melatonin group; C ¼ control group.
other antioxidants, melatonin dissolves both in water and in lipids; therefore, it reaches all cell components, where it can act as an antioxidant. In brain ischemia and reperfusion models, melatonin has been shown to decrease damage [10–13, 19, 20]. These changes were attributed to the antioxidative capacity of melatonin. In human studies, melatonin has been given in a very large range of doses, from 0.1 mg to 2000 mg. The large doses are consistent with animal studies, demonstrating the very low toxicity of this hormone, even at pharmacologic doses; doses over 0.5 mg are accepted as TABLE 4 Statistical Results of the Electrophysiological Study in Group Comparisons (Mann-Whitney U test) Grup 1(P)-2(M) -3(C) (n ¼ 24) Latency (ms)
Amplitude (mV)
Nerve conduction velocity (m/ms)
P-M P-C M-C P-M P-C M-C P-M P-C M-C
P values before nerve repairing
P values at 12th week
P ¼ 0.110 P ¼ 0.873 P ¼ 0.143 P ¼ 0.400 P ¼ 0.753 P ¼ 0.834 P ¼ 0.170 P ¼ 0.423 P ¼ 0.332
P ¼ 0.022 P ¼ 0.526 P ¼ 0.023 P ¼ 0.008 P ¼ 0.713 P ¼ 0.009 P ¼ 0.004 P ¼ 0.072 P ¼ 0.006
P ¼ pinealectomy group; M ¼ melatonin group; C ¼ control group.
pharmacologic [8, 21]. Melatonin displays its antioxidant effects by means of two mechanisms. The first is through its direct influence on toxic radicals, and this is independent of receptors. This feature depends upon its strong binding of hydroxyl and peroxyl radicals. The second effect is through the receptors over the genomes. Through these receptors, it induces enzymes that detoxify free radicals [10–22]. However, both the direct and indirect effects do not occur sufficiently at physiologic levels. It has been suggested that the antioxidant effects can be seen more during the significantly higher concentrations associated with nocturnal peak serum concentrations [8]. Melatonin has many other known effects that pertain to our study hypothesis other than its antioxidant effect [10, 11]. In experimental models, intracellular calcium levels have a central role in nerve recovery. Venacek et al. and Angelov et al. have shown that melatonin prevents intracellular calcium influx by influencing voltage-dependent calcium channels, thereby mimicking the effects of Ca channel blockers [23, 24]. Based upon the above-mentioned information, we know that even if rapid and successful repair nerve is achieved, complete functional restoration is not feasible. Too many inevitable factors prevent complete recovery. One of these is disruption of intraneural microcirculation and the subsequent development of ischemia along the repair line. During ischemia, the concentration of calcium ions increases in the cytosol, cell swelling develops, and toxic free radicals increase in number, all of which lead to neural tissue damage. We propose that melatonin exerts its nerve protection effect by preventing intracellular calcium influx and free radical-induced damage. In this study, we evaluated the effects of melatonin on nerve healing in several ways: functionally, electrophysiologically, and histologically. Histologic evaluation revealed prominent improvement in the melatonin group, demonstrating melatonin’s positive effect on nerve tissue repair. However, even though there was marked improvement in the control group, there was no significant improvement relative to the pinealectomy group. Weichselbaum et al. demonstrated that in pinealectomized rats, wound healing is slower than in controls, and in rats given melatonin, faster [25]. Drobnik et al. found that pinealectomy increased the amount of collagen, whereas melatonin, given in doses of 30–100 ug, decreased the collagen amount [26]. These findings are consistent with our findings. In our study, the amount of collagen laid down was greatest in the pinealectomy group and its organization was worst. In controls, a nonsignificant decrease in collagen amount was noted; but rats that received melatonin had significantly less and better-organized collagen. Even with a technically excellent nerve repair,
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scar formation is inevitable. Inhibition of collagen formation contributes meaningfully to nerve recovery. These findings show us that melatonin has little effect in physiologic doses. The desired effect of melatonin is achieved only when it is given in high enough doses, whether that by local or systemic administration. The second segment of our study involved a functional evaluation, which consisted of a walking track analysis. Selection of appropriate evaluation methods is crucial when measuring experimental nerve recovery. In 1982, De Medinaceli first reported that the SFI could be used to evaluate total lower limb function, including nerve, muscle, and joint function in rats [27]. Since then, many investigators have used walking track analysis as an assessment of global function recovery after sciatic nerve injuries or repairs. The use of walking track analysis provides a noninvasive method by which to assess the functional status of the sciatic nerve, avoiding interaction with the regeneration process, based on measurement of footprints, expressed in units of functional deficit. Because proper walking requires coordinated functions, including sensory input, motor responses, and cortical integration, SFI may be better than extrapolating from the basic electrophysiology and histomorphometry of axon growth and muscle innervation, especially if the research focus concerns functional outcome [28]. In our study, the results of walking track analysis supported the histologic findings. This analysis revealed significant improvement in the melatonin group, but no significant difference between controls and rats that had undergone a pinealectomy, again showing us that a desirable effect can only be achieved with melatonin doses producing higher than physiologic levels. The third and final part of our study was an electrophysiologic evaluation. In this evaluation, the only inter-group difference and difference versus baseline occurred in the melatonin group at 12 wk data collection point, suggesting again that melatonin is effective only when given in supraphysiologic doses; moreover, it needs to be given over a relatively long period. Combining these findings together, we conclude that melatonin has a significant beneficial effect on nerve recovery, that this effect is apparent functionally, histopathologically and electrophysiologically, and that the results observed in each of these three areas appear to be consistent. However, for these effects to occur, the dose of melatonin must be high enough to achieve supraphysiologic levels of the hormone.
CONCLUSIONS
Peripheral nerve injuries continue to be a major clinical challenge, so that the search for better treatments
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and a better understanding of nerve injury and repair is ongoing. Several surgical techniques and medical agents have been tried, in an attempt to establish some consensus as to the most effective treatment protocol. Our study provides a novel insight into this unclear field, in that it illustrates melatonin’s potential as a nerve reparative agent. The beneficial effects of melatonin cannot be achieved at physiologic doses, however. Higher doses and a longer duration of administration are necessary. Future research is warranted to compare melatonin’s effects versus those of other nerve-regenerating agents, and ultimately to explore the effectiveness of melatonin in human nerve injury patients within the context of a randomized, doubleblinded clinical trial. REFERENCES 1. Brushart TM. Nerve repair and grafting. In: Green DP, Ed. Operative hand surgery. 4th ed. Edinburg: Churchill Livingstone, 1999. p. 1381. 2. Terzis JK, Smith KL. Repair and grafting of the peripheral nerve. In: McCarthy JG, Ed. Plastic surgery. Philadelphia: WB Saunders, 1990. p. 630. 3. Sunderland S. The history of nerve repair. A critical appraisal. Edinburgh: Churchill Livingstone, 1991. 4. Lundborg G. Nerve regeneration and repair. A review. Acta Orthop Scand 1987;58:145. 5. Sunderland S. Nerves and nerve injuries. Edinburgh: Churchill Livingstone, 1978. 6. Myers RR, Yamamoto T, Yaksh TL, et al. The role of focal nerve ischemia and Wallerian degeneration in peripheral nerve injury producing hyperesthesia. Anesthesiology 1993;78:308. 7. Kater SB, Mills LR. Regulation of growth cone behavior by calcium. J Neurosci 1991;11:891. 8. Brzezinski A. Melatonin in humans. N Engl J Med 1997;336:186. 9. Cagnacci A. Melatonin in relation to physiology in adult humans. J Pineal Res 1996;21:200. 10. Pierrefiche G, Zerbib R, Laborit H. Anxiolytic activity of melatonin in mice: Involvement of benzodiazepine receptors. Res Commun Chem Pathol Pharmacol 1993;82:131. 11. Antolin I, Rodriguez C, Sainz RM, et al. Neurohormone melatonin prevents cell damage: Effect on gene expression for antioxidant enzymes. FASEB J 1996;10:882. 12. Pieri C, Marra M, Moroni F, et al. Melatonin: A peroxyl radical scavenger more effective than vitamin E. Life Sci 1994; 55:PL271. 13. Poeggeler B, Saarela S, Reiter RJ, et al. Melatonin—A highly potent endogenous radical scavenger and electron donor: New aspects of the oxidation chemistry of this indole accessed in vitro. Ann N Y Acad Sci 1994;738:419. 14. Pohlymeyer G, Reuss S, Baum A. An improved technique for visually controlled pinealectomy in the rat. J Exp Anim Sci 1994; 36:84. 15. Komiyama A, Novicki DL, Suzuki K. Adhesion and proliferation are enhanced in vitro in Schwann cells from nerve undergoing Wallerian degeneration. J Neurosci Res 1991;29:308. 16. Lundborg G, Longo FM, Varon S. Nerve regeneration model and trophic factors in vivo. Brain Res 1982;232:157. 17. Van der Zee CE, Brakkee JH, Gispen WH. Putative neurotrophic factors and functional recovery from peripheral nerve damage in the rat. Br J Pharmacol 1991;103:1041. 18. Lipton SA. Growth factors for neuronal survival and process regeneration. Implications in the mammalian central nervous system. Arch Neurol 1989;46:1241.
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