REVIEW ARTICLE: Melatonin plus exercise-based neurorehabilitative therapy for spinal cord injury
Address reprint request to Yonggeun Hong, Department of Physical Therapy, Cardiovascular & Metabolic Disease Center, College of Biomedical Science & Engineering, Inje University, 607 O-bang Dong, Gimhae 621-749, Korea.
Kyu-Tae Chang, National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Ochang 363-883, Korea.
Abstract: Spinal cord injury (SCI) is damage to the spinal cord caused by the trauma or disease that results in compromised or loss of body function. Subsequent to SCI in humans, many individuals have residual motor and sensory deficits that impair functional performance and quality of life. The available treatments for SCI are rehabilitation therapy, activity-based therapies, and pharmacological treatment using antioxidants and their agonists. Among pharmacological treatments, the most efficient and commonly used antioxidant for experimental SCI treatment is melatonin, an indolamine secreted by pineal gland at night. Melatonin’s receptor-independent free radical scavenging action and its broad-spectrum antioxidant activity makes it an ideal antioxidant to protect tissue from oxidative stress-induced secondary damage after SCI. Owing to the limitations of an activity-based therapy and antioxidant treatment singly on the functional recovery and oxidative stress-induced secondary damages after SCI, a melatonin plus exercise treatment may be a more effective therapy for SCI. As suggested herein, supplementation with melatonin in conjunction with exercise not only would improve the functional recovery by enhancing the beneficial effects of exercise but would reduce the secondary tissue damage simultaneously. Finally, melatonin may protect against exercise-induced fatigue and impairments. In this review, based on the documented evidence regarding the beneficial effects of melatonin, activity-based therapy and the combination of both on functional recovery, as well as reduction of secondary damage caused by oxidative stress after SCI, we suggest the melatonin combined with exercise would be a novel neurorehabilitative strategy for the faster recovery after SCI.
SCI is damage to the spinal cord that results in a loss of function such as mobility or feeling. The common causes of damage are trauma (car accident, gunshot, falls, etc.) or disease (polio, spinal bifida, Friedreich’s ataxia, etc.). SCIs can occur at any level of the cord, and they compromise or cause loss of body function which is specifically associated with the segment of the cord that is injured and the severity of the injury. Because the spinal cord acts as the main information pathway between the brain and the rest of the body, a SCI can have significant physiological consequences. Following SCI in humans, many individuals have residual motor and sensory deficits that impair functional performance and quality of life. Dependence on a wheelchair for mobility and development of neuropathic pain are two of the most limiting and most common impairments after SCI [1–3].
It is well documented that the pathophysiology of SCI involves a two-step process, with primary and secondary mechanisms. The primary traumatic mechanical injury to the spinal cord causes death of a number of neurons . These events are then exacerbated by a variety of secondary mechanisms including vascular changes, ischemia, vasospasms, hemorrhage and thrombosis, neurotransmitter (especially glutamate) accumulation, generation of free radicals and nitric oxide (NO), calcium overload, compromised energy metabolism and inflammatory factors [5, 6]. The secondary damage is determined by many cellular, molecular, and biochemical cascades. This active and progressive spread of damage results from a process that begins within minutes and continues for weeks after the initial injury.
Increased production of reactive oxygen species also appears to play a key role in the induction of neurological dysfunction in the course of SCI . Oxidative stress is known to cause damage to lipids, proteins, and nucleic acids, which may alter cellular function and result in cytotoxicity and tissue damage . Additionally, enhanced molecular oxidation activates redox-responsive transcription factors and thus stimulates inflammatory reactions in the injured spinal cord . Although a large numbers of researchers and medical practitioners have worked on SCI using a variety of experimental animal models and clinical trials, the questions related to the mechanisms involved in SCI and its secondary damage remain unanswered.
The process of inflammation after SCI is initiated by glial cells and T cells that activate macrophages to mount an immune response. Inflammation is the physiologic process by which vascularized tissues respond to injury; it is characterized by fluid accumulation and the influx of plasma proteins, neutrophils, T lymphocytes and macrophages. Many cytokines, including tumor necrosis factor (TNF) and interleukins 1, 6 and 10, are released during the secondary immune reaction mediated by the macrophages and microglia. These cytokines induce the activation of other substances such as chemokines and NO and further aggravate the secondary immune reaction.
Rehabilitation programs combine physical therapies with skill-building activities and counseling to provide social and emotional support by educating the injured person and his/her family and friends. Neurorehabilitation is a complex medical process that aims to assist recovery, to minimize and/or compensate for any serious disability (such as that caused by a severe spinal injury or brain damage) and functional alterations resulting from nervous system injury. Different neurorehabilitative strategies that are being used for promoting plasticity and recovery from CNS injury are passive exercise, active modes of exercise, and neuroprostheses for electrical activation of motor neurons and sensory afferents. Neurorehabilitation strategies focus mainly on promoting the capacity for continuous alterations of the neural pathways and synapses of the nervous system for regaining function and repairing the damaged connections caused by injury. The rehabilitative strategies help in re-establishing supraspinal control of caudal circuitry through novel supraspinal-spinal circuits by promoting rewiring of the cortex to bypass pathways interrupted owing to an incomplete SCI . Because SCIs are heterogeneous in casualty, severity, and location of injury, designing an effective common rehabilitation strategy has become an incredibly challenging task to people involved in medicine and basic research.
Melatonin and its metabolites possess several properties that may be responsible for their neuroprotective effects; especially noteworthy are their abilities to scavenge free radicals [11–17]. Melatonin is highly effective in reducing the oxidation of lipids, proteins, and DNA [18–20]. These effects may be because melatonin itself is a powerful antioxidant and moreover, it also stimulates antioxidant enzymes including catalase, glutathione reductase, glutathione peroxidase (GPx), and superoxide dismutase (SOD) . Melatonin also reportedly attenuates glutamate-mediated Ca2+ influx and inflammation by inhibiting the levels of pro-inflammatory cytokines [22–26]. The apoptosis reducing ability of melatonin in brain cells and in other tissues is also well documented [27–31].
The capacity of repetitive locomotor activity to promote functional restoration after CNS injury is well recognized [32, 33]. Although neurotrophins have been identified as molecular systems with the potential to enhance spinal cord repair, most of the strategies to induce motor recovery after SCI have involved the addition of exogenous neurotrophins into the CNS. Given the capacity of voluntary exercise to modulate endogenous neurotrophins in the spinal cord , many studies have shown the potential of motor training to promote functional recovery after SCI , although the specific mechanisms and molecular systems involved remain largely unidentified. Factors produced by exercised muscles that may influence the spinal cord and neurotrophic factors are potential candidates for mediating these effects . Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) are known to be elevated in muscles that have been subjected to increased activity [36, 37] and can be retrogradely transported to the spinal cord. BDNF is transferred between synapses in an activity-dependent manner, and it may therefore influence neurons distant from the site of entry into the cord .
We have observed an improved motor function recovery that involves an anti-inflammatory response and neuromuscular integration following a combined therapy of exercise and melatonin after SCI in rats . Motor neuronal degeneration and locomotor activity caused by the local upregulation of inducible nitric oxide synthase (iNOS) following the administration of melatonin and exercise after spinal cord contusion injury in male rats was reported. Attenuation of iNOS after melatonin treatment was observed in parallel with a delayed progression of the injury resulting in a significantly improved functional outcome .
Rehabilitative therapy for SCI focuses on passive exercise at early stages of postinjury to promote plasticity caudal to injury and mitigate the spasticity followed by active exercise to enhance plasticity both rostral and caudal to injury . Although voluntary exercise is a promising therapy for promoting the recovery and activity-dependent plasticity throughout the neuraxis after incomplete SCI, this can be performed only by the individuals having some motor functions . Hence, there is a great demand for simple, multifaceted treatment to maximize the effectiveness of neurorehabilitative therapies for SCI. In our laboratory, we have been working on experimental SCI with multiple therapy approaches to develop an animal model for appropriate neurorehabilitative strategy for SCI. Thus, the purpose this report is to review the research carried out on the synergistic effect of exercise combined with melatonin treatment to enhance the knowledge required to develop an animal model for neurorehabilitation therapy for SCI.
Melatonin is a widely distributed neurotransmitter-like compound/indoleamine produced especially at night in the pineal gland . Its secretion occurs during darkness and inhibited by light. It is also produced in a number of other areas, for example, the gastrointestinal tract [40–42]. Chemically, melatonin is N-acetyl-5-methoxytryptamine, a derivative of serotonin, which in turn is derived from the amino acid tryptophan. Serotonin is first acetylated by N-acetyltransferase (probably the rate-limiting step) and then methylated by hydroxyindole O-methyltransferase to form melatonin . Melatonin synthesis depends on intact beta-adrenergic receptor function on the pinealocytes . Norepinephrine activates the N-acetyltransferase, while beta-receptor blockers depress melatonin secretion . Melatonin occurs in animals as well as plant, bananas, beets, cucumbers, and tomatoes [46–50]. Melatonin was first discovered in connection with the mechanism by which some amphibians and reptiles change the color of their skin [51, 52]. Lynch et al.  have demonstrated the production and involvement of melatonin in circadian rhythms in human. Membrane melatonin receptors are widely distributed including being present in the suprachiasmatic nuclei (SCN) of the hypothalamus or melatonin may act directly on SCN to influence circadian rhythms. Because, it plays an important role in several aspects of circadian rhythms, melatonin is often called a chronobiotic .
Melatonin is released from the pinealocytes into the blood and also into the cerebrospinal fluid in the third ventricle of the brain . In addition to being found in the blood and cerebrospinal fluid, melatonin is present in other fluids as well , often in higher concentrations than measured in the blood.
Melatonin, either endogenously produced or exogenously supplemented, is a potent indirect antioxidant, via its stimulatory actions on antioxidative enzymes [56–58], and a direct free radical scavenging activity . Melatonin also has oncostatic, neurorprotective, anti-inflammatory and immunomodulatory effects [29, 60]. Free radicals are chemical constituents that have an unpaired electron. The addition of an electron to O2 leads to formation of superoxide anion radical (). The is dismutated by superoxide dismutase to H2O2. It is rapidly converted to hydroxyl radical (˙OH) via the Fenton reaction in the presence of transition metals. ˙OH is a highly reactive free radical, which damages essentially all molecules that contributes to neurodegenerative, cancer, and autoimmune diseases [58, 61–65]. Melatonin is an efficient scavenger of the ˙OH and other toxic derivatives of O2 .
The receptor-independent free radical scavenger and a broad-spectrum antioxidant activity have stimulated interest in the use of melatonin in experimental and clinical situations [12, 67]. Beyond its multifaceted functions and various biological effects, melatonin has low toxicity  and is available in a pure form and at low cost . This combination features makes it an ideal antioxidant to protect, against tissue damage caused by oxidative stress during various pathophysiologic processes as well as for a range of clinical and wellness-enhancing applications.
Effect of melatonin on SCI
In humans, numerous beneficial effects of both endogenously- and exogenously administered melatonin have been reported in several health disorders including insomnia, osteoporosis, postmenopausal sleep disorder, depression and anxiety, breast cancer, prostate cancer, rheumatoid arthritis, and as a short-term hypnotic for schizophrenic patients with insomnia.
In addition to neutralizing free radicals, melatonin plays an important role in activating the enzymatic defense system by inducing the activation of several antioxidant enzymes including SOD, GPx, glutathione reductase (GRd), and glucose-6-phosphate dehydrogenase [69–74]. Melatonin directly neutralizes the ˙OH via a nonenzymatic reaction without involving a membrane receptor: in this context melatonin was more efficient than glutathione or mannitol [71, 75]. In a process known as radical avoidance, melatonin diminishes free radical generation by preventing electron leakage from the mitochondrial electron transport chain [76–78].
Erol et al.  reported that melatonin was significantly better than octreotide in preventing the congestion, edema, axonal degeneration and necrosis in experimentally induced SCI in rats. Histological studies showed a marked reduction in necrotic cell death and degeneration in both the initial and late stages of SCI in experimentally injured rats treated with melatonin. Octreotide-treated rats also showed a significant reduction in necrosis and degeneration only during the late stages but did limit edema and congestion in both the initial and the late stages of injury . They concluded that melatonin is superior to octreotide in protecting the spinal cord from further injury.
The beneficial effects of melatonin in decreasing the cyst formation and tissue healing relate to its ability to reduce edema and inflammation via the prevention of adhesion molecule production and leukocyte adhesion to endothelial cells [56, 70]. The efficacy of melatonin as a neuroprotective agent at the level of the spinal cord has been observed in a variety of experimental situations [70, 80–82].
Melatonin is known to play an important role in attenuating glutamate-mediated Ca2+ influx and inflammation by inhibiting the levels of pro-inflammatory cytokines [22–24]. In addition, the multifaceted properties of melatonin have documented its ability to inhibit apoptosis of brain cells and in other tissues as well [83–85] and in injuries associated with the CNS in animals [23, 60, 86, 87].
Samantaray et al.  demonstrated that melatonin inhibited inflammation by reducing the activation of astrocytes, microglia, and macrophages and reduced axonal degeneration following SCI injury. In addition, they also reported that melatonin’s attenuation of neuronal death was accompanied by a significant reduction in calpain expression and caspase-3 activity in acute SCI rats. These findings are consistent with the results of a publication by Genovesev et al.  who reported that melatonin treatment significantly improved the recovery of limb function (evaluated by motor recovery score) and had potent anti-inflammatory effects in an animal model of SCI.
In behavioral studies, chronic melatonin treatment was found to significantly improve spatial memory in the radial-maze and motor coordination in rota-rod test . Melatonin also significantly reduced SCI-induced tissue alterations including the degree of histological damage, phospho-p38 expression, phospho-JNK expression, phospho-ERK1/2 and phospho-ERK2 expression, pro-inflammatory cytokine production, and expression of high mobility group box (HMGB)-1 protein and improved motor function in mice 24 hr after experimental SCI . This group feels that the ability of melatonin to limit SCI damage probably is linked to the reduction of MAPK signaling pathways and HMGB1 expression in mice.
The activation of matrix metalloproteinase (MMP)-2 and MMP-9 disrupts the blood–brain/spinal cord barrier and promotes inflammation that leads to early secondary pathogenesis in these tissues [90–92]. A single publication has reported that melatonin reduced the activity and expression of MMP-9 and MMP-2 in an experimental model of SCI in mice .
Because of its high solubility, lipophilicity, ability to cross morphophysiological barriers (blood–brain barrier, cell membrane, and basal membrane), and diffuse into all body fluids, exogenous administration of melatonin provides on-site protection against free radical damage [59, 72]. In experimental rat models of SCI (drop weight method), functional recovery was incomplete even after 6 wk of injury in untreated animals , but almost complete recovery was appeared in melatonin-injected rats within 3 wk after injury . This protective action of melatonin was attributed to its ability to scavenge free radicals.
Apart from well-documented antioxidant function of melatonin, the indoleamine also facilitates the recovery of damaged spinal cord by reducing the occurrence of neutrophil-induced lipid peroxidation, thiobarbituric acid reactive substances, and elevated myeloperoxidase activity in experimental model injury to the cord of mice . Erten et al.  also have reported protective effects of both pre- and postinjury melatonin administration in an ischemia-reperfusion injury model of the spinal cord of rabbits. In this study, exogenously administered melatonin significantly enhanced the activities of endogenous antioxidant enzymes in the damaged tissue. Activated antioxidative enzymes convert radical products to innocuous molecules and reduce the possible damages due to free radical toxic agents . The neuroprotective effects of melatonin in neurons, myelin, axons, and subcellular organelles including the nucleus and mitochondria are also apparent during ultrastructural studies [80, 81].
Neurorehabilitation is a complex medical process that aims to assist recovery, to minimize and/or compensate for any functional alterations or serious disability resulting from nervous system injury. Different neurorehabilitative strategies are being used to promote plasticity and recovery form CNS injury. These include passive exercise, active exercise, and neuroprostheses for electrical activation of motor neurons and sensory afferents. In this review, we will concentrate mainly on the active exercise alone or in combination with melatonin as a rehabilitative strategy for SCI. The active exercise approach involves the routine assisted/unassisted active movements using varying degrees of supraspinal and/or segmental spinal control. Multiple active exercise approaches including locomotor training (manual-assisted and robot-assisted partial weight-supported treadmill training, as well as overground locomotion), repetitive upper-limb training, and general exercise/environmental enrichment are recommended to treat patients with incomplete SCIs.
Effect of exercise on SCI
Emerging evidence suggests that increased physical activity associated with a dynamic lifestyle not only reduces the risk of diseases such as obesity, cardiovascular diseases, type 2 diabetes, osteoporosis, cancer, and depression [95–97] but also protects from several neurological diseases including Parkinson’s disease , Alzheimer’s dementia , and ischemic stroke .
Active exercise mediates plasticity at multiple levels of the neuraxis including the cortex, descending supraspinal motor pathways, and spinal cord circuitry caudal to injury . Voluntary exercise improves functional joint motion and also induces functional activation of muscles and multiple modes of afferent stimulation. Although there is a lack of knowledge on the role and degree of specificity of locomotor training needed to achieve extensive recovery, improved postinjury motor recovery was reported following locomotor training . The reduced expression of inhibitory molecules and enhanced expression of neurotrophic factors along with alterations in electrophysiological properties in the lumbar cord segment following locomotor training has been reported [33, 102–104] in both rodent and feline models of thoracic SCI. Studies conducted in spinal cord-transected cats reported task-specific and sensory feedback mechanisms dependent recovery after locomotor training [105, 106]. Studies also showed partial recovery of hind limb locomotion [32, 33, 103, 107, 108] and improved sensation  in exercise and treadmill-trained rodents as well as improved recovery in humans following overground and partial body weight-supported treadmill training in incomplete SCIs [109–120]. Moreover, enhanced locomotor function by improved corticospinal drive to muscle of the lower limb was observed among treadmill-trained humans after incomplete SCI [121, 122].
Information on the substrates and implications of activity-dependent cortical reorganization in cortical plasticity after SCI is limited. However, several studies have noted activity-dependent reorganization of motor cortex (cortical motor neurons) in neurologically intact nonhuman primates  and cortical map in humans following cyclic intensive training after a cervical spinal injury and robotic locomotor training after a thoracic spinal injury [124–126]. Several publications have reported a possible role of voluntary or differential voluntary exercise regimens in facilitating neuroplasticity, neuronal growth, and functional recovery or differential recovery through upregulation of expression of BDNF in the skeletal muscles of rats [35, 127–129]. These studies clearly demonstrated the occurrence of neuroplasticity through BDNF-mediated pathways after SCI [34–36, 130, 131].
Besides the increase in expression of genes that encode several brain neurotrophins (e.g. BDNF) [132–134], nerve growth factor [133, 135], and galanin  in persistently active rats and mice, exercise also significantly elevated levels of mitochondrial uncoupling protein 2 , which appears to promote learning and memory by modulating the BDNF production by hippocampal cells and molecular systems downstream of BDNF action .
Synergistic effect of melatonin on SCI
At a concentration of 50 mg/kg, melatonin exerted protective effects in a rat model of SCI, whereas the melatonin or dexamethasone was ineffective when administered as single treatment at concentrations of 10 and 0.025 mg/kg, respectively [82, 138]. Anti-inflammatory actions were not noticed in animals treated with melatonin (10 mg/kg) or dexamethasone (0.025 mg/kg) alone, whereas the combination therapy exerted a strong beneficial effect in reducing the tissue damage, the degree of neutrophil infiltration at different time points, TNF-α expression, the nitration of tyrosine residues, iNOS expression, degree of apoptosis, and motor recovery in experimental model of SCI in mice . The reduction in the degree of secondary damage associated with SCI following combined therapy with melatonin and dexamethasone strongly suggests the potential of melatonin in conjunction with steroids to reduce the dose and of side effects associated with steroid treatment for inflammatory conditions .
Although it is a well documented that steroids have several beneficial effects including disease-modifying, anti-inflammatory actions and on acute CNS injury in humans and experimental animals [139, 140], the main concern regarding the use of steroids in clinical applications is their possible long-term effects that lead to a series of unwanted side effects on hypothalamus-pituitary-adrenal axis, the cardiovascular system, as well as on fat and bone metabolism [83, 141].
Cayli et al.  performed a study to clarify the synergistic effects of melatonin and methylprednisolone (MP) on neurological recovery after experimental SCI in albino rats. Although the combined action of MP and melatonin was elevated in terms of the degree of lipid peroxidation, there were no significant differences in the effects of melatonin or MP alone or in combination with the neurobehavioral, electrophysiological, and ultrastructural recovery on the 10th day after experimental SCI in rats . This enhanced biochemical recovery following the combined treatment may have been the result of the cumulative effects of the different antioxidative mechanisms of MP and melatonin at the site of cord injury.
De Leon and Acosta  performed a study to evaluate the effect of chronic quipazine in combination with robotic-assisted treadmill training on the hind limb stepping in experimental spinal-transected rats. Quipazine [2-(1-piperazinyl)-Quinoline] is a serotonin agonist, which is widely used in pharmacological applications as an oxytocin and antidepressive agent. They found enhanced hind limb stepping on the treadmill among the robotic-assisted training rats but not in the untrained animals after experimental spinal transection. In contrast to previous observations  using exercise and melatonin, they did not find a significant effect of combined therapy of robotic-assisted treadmill training and quipazine on hindlimb stepping in rats after experimental spinal transection  compared to rats that received only the robotic-assisted treadmill training.
In an experimental animal model of SCI, the administration of melatonin in combination with exercise documented significantly increased hind limb movement, a reduced level of iNOS mRNA and an increased number of motor neurons in the ventral horn compared to control SCI and SCI plus exercise alone rats . In contrast, Cayli et al.  reported the beneficial synergistic effects of two antioxidants, melatonin and MP only in terms of a reduction in lipid peroxidation.
Based on the observations of our previous study on the synergistic effect of melatonin and exercise on SCI, we have hypothesized that combined therapy of melatonin and exercise would improve motor function recovery, via an anti-inflammatory actions and neuromuscular integration in rats. The marked reduction in the degree of secondary damage associated with SCI in rats treated with combined with melatonin and exercise strongly suggests that exercise in combination with melatonin administration may be an important rehabilitative strategy to treat SCI as well as to reduce the side effects related to exercise-induced fatigue and impairment.
To date, most animal model and human studies on the recovery of function after SCI have focused on a various strategies of treatment including (i) passive exercise, (ii) active exercise, (iii) a combination of passive and active exercise, (iv) the use of antioxidants singly or in combination, (v) steroids treatment, and (vi) steroids in combination with antioxidants. To our knowledge, there are no published reports on the cumulative effects of activity-based therapy in conjunction with either antioxidant or steroid therapy on SCI either in experimental animal models or human clinical studies. We have been interested in determining the combined effect of melatonin and exercise on the SCI using an experimental animal model. Recently, we reported on the synergistic effect of melatonin on exercise-induced neuronal reconstruction and functional recovery in a SCI animal model . Currently, we are investigating the effect of melatonin in combination with exercise on SCI with different therapeutic modalities in an experimental animal model. We also suggest the further systematic investigation into the effect of melatonin and other antioxidants in combination with exercise on the recovery of SCI as a novel neurorehabilitative strategy for SCI.
The knowledge gained from findings of the systematic investigation into the neurorehabilitative strategies in animals will provide a foundation for novel similar therapies in humans suffering with injuries to their spinal cord. Evidence from the basic scientists will be translated into therapeutic guidelines and incorporate into novel neurorehabilitative treatments to promote the functional recovery from SCI.
This work was supported by grant (Code No. 20100301-061-100-001-03-00 to Y. Hong) from BioGreen 21 Program, Rural Development Administration. Republic of Korea.