Spinal cord injury
Clinically, SCI involves two components: an initial mechanical instability precipitating into a secondary injury process leading to the final neurological deficit . Traumatic SCI may be a direct lesion that causes focal injury to the neural elements at the site of impact or a lesion due to stretching or compressive forces applied to spinal cord via bones and/or ligaments. In either case, the cord lesion expands with time following injury over adjoining spinal segments causing secondary injury. Primary injuries cause disruption of the blood supply to spinal cord and lead to ischemic damage. All spinal cord lesions can be broadly classified as neurologically complete or incomplete by the classical Frankel score  or using the more recent ASIA scale . However, regardless of the mode of induction of the primary injury, SCI essentially evolves through an acute and chronic phase as inferred by a substantial body of evidence [3, 40, 41]. We provide a schematic representation of the sequence of events that occur following a traumatic SCI (Fig. 1).
Experimental SCI induction is largely guided by the objective of the investigator. Animal models of SCI may include contusion, compression, or transection. The contusion model is the choice to study physiologic responses whereas a clip or balloon compression model is preferred to study the effects of compression or the optimal timing of decompression . However, to test the implementation of a device, a partial or complete transection model is preferred. A combination of models might also be an option such as initial transection mode at early stages of an experimental plan to explore axonal regeneration followed by testing the most promising therapy in clinically relevant contusion models. In our laboratory, for the past three decades, we have been using a clinically relevant contusion model to induce moderately severe injury using the weight drop method .
Although complete recovery after SCI is yet to be attained, research in the field over the past several decades has definitely boosted the belief of the researchers as well as the survivors in the possibility of attaining greater motor function. A recent review by Akhtar et al. (2008) has rationally pin-pointed the shortcomings in loosing the translation of experimental SCI research from bench to bed-side as the discrepancies in several aspects between the animal models and clinical trauma; these include anatomical localization, laminectomy, anesthesia, laboratory stressors, surrogate markers for behavior analysis in experimental SCI, and functional assessment scales (sensory and motor) . Overcoming these pitfalls while designing the experimental SCI will certainly amplify the translational potential of experimental SCI research. Several ongoing patents on plausible SCI therapeutic interventions have been enlisted in recent publications. However, like other fields of traumatic injuries in the CNS, encouraging results obtained in experimental models of SCI have so far failed to identify any safe and effective neuroprotectants in clinical trials as confirmed recently by the International Campaign for Cures of Spinal Cord Injury and Paralysis . A quest for a good neuroprotectant in SCI remains unresolved. Hence, a mini review on melatonin’s efficacy in the treatment of SCI is timely.
Melatonin efficacy in SCI
A possible correlation between the neuroprotective efficacy of melatonin and SCI originally emerged almost three decades ago as clinical case reports in which the circardian profiles of endogenous melatonin were assessed in SCI patients. Much later the experimental studies on the utility of exogenously-applied melatonin in experimental models of SCI were conducted. Hence, in the following discourse the outcomes of initial clinical case reports are considered first followed by a discussion of the results of the subsequent experimental studies.
Levels of melatonin in SCI patients differed strictly with respect to the site of injury. SCI disrupting the cervical spinal cord significantly perturbed the levels of endogenous melatonin, whereas, an injury at the levels of thoracic or lumbar did not affect it severely. These differences are explicable in terms of the central neural pathways, which connect the eyes to the pineal gland. The first clinical study revealed that lesions within the cervical spinal column caused decentralization of pineal gland because they perturbed descending sympathetic fibers and led to an absence of significant increment in nocturnal melatonin; this, clearly distinguished quadriplegic subjects from normal males and from the subject with a lesion of the lumbar spinal cord . More than two decades later, it was further confirmed that neurologically complete cervical spinal cord transection results in complete loss of the circardian melatonin rhythm . More complete studies on rhythms of serum melatonin in patients with spinal lesions at the cervical, thoracic, or lumbar region revealed that the cervical region of the spinal cord includes the neural pathway, which is essential for the diurnal rhythm of pineal melatonin secretion in human beings. Retrospectively, such studies clearly discerned the relationship between regional specificity of SCI lesions and changes in the endogenous profile of melatonin . Such a regional bias was re-confirmed in a recent clinical study conducted using tetraplegic patients with bilateral oculosympathetic paresis showing a complete loss of the nocturnal production of melatonin .
A recent assessment based on the ‘Basic Nordic Sleep Questionaire’ conducted at the Karolinska University Hospital in Stockholm, on a large SCI patient population comprised of 230 patients, inferred that perturbances in the melatonin rhythm contributed to poor subjective sleep quality associated with higher ratings of pain intensity, anxiety, and depression . A year later, another study confirmed the positive correlation of an altered melatonin cycle in cervical SCI patients with reduced sleep quality and documented the need for larger studies on the potential usefulness of melatonin replacement therapy in normalizing sleep in SCI patients .
Although the field is in its infancy, several encouraging clinical case studies suggest that melatonin may be the neuroprotectant of choice in this devastating injury. It has already been mentioned earlier that there is no neuroprotective drug in clinical practice to date that is highly useful as a treatment for this complex neurological problem. Hence, it is important to assess the outcomes of melatonin’s use in experimental models of SCI with the intent of eventually applying this information for treatment of SCI in humans.
The first experimental study to test melatonin’s efficacy in reducing neural damage in experimental SCI in animals is that of Fujimoto and colleagues . They studied an acute to chronic SCI model and showed a significant protection by melatonin against neutrophil-mediated damage including lipid peroxidation, reduced levels of secondary injury, and an earlier recovery . Systemically applied pharmacological doses of melatonin were shown to boost the anti-oxidant defense system in a variety of ways after acute SCI . Melatonin also protected against autodestruction following SCI by reducing the levels of free iron and the products of lipid peroxidation .
When compared with methylprednisolone, melatonin was found to be more effective in an acute SCI model against lipid peroxidation and in preserving the structural integrity of neurons, axons, and intracellular organelles . A later study uncovered the greater efficacy of melatonin compared to methylprednisolone in preserving the ultrastructural histopathological integrity of the spinal cord although the reduction of lipid peroxidation after SCI was less obvious. Furthermore, this study also verified that the neuroprotective effect of melatonin was dose-dependent . A combination therapy of melatonin with methylprednisolone did show an additive effect against the accumulation of lipid peroxidation products in the subacute phase of injury, but did not show any difference in a brief chronic 10-day study following SCI in rats as evidenced by neurobehavioral, ultrastructural, and electrophysiological recovery . However, more recently in a SCI model in mice, the combination therapy with melatonin and dexamethasone had a significant and important beneficial anti-inflammatory effect by blocking the possible progression of secondary injury events after SCI. This study showed that the effective dose of dexamethasone could be reduced 10-fold when it was given in combination with melatonin .
In comparision to other anti-oxidants such as oxytetracycline or prostaglandin E1, melatonin was found to be more potent in ameliorating secondary damage in an acute model of SCI . Likewise, melatonin was more effective than octreotide, an octapeptide that mimics natural somatostatin pharmacologically and is known to reduce lipid peroxidation as well as curtailing the degree of edema and other consequences of ischemia . Melatonin reduces the development of inflammation and tissue injury associated with SCI by blocking both oxidative and nitrosative stress . Moreover, melatonin limits the expression and activity of matrix metalloproteinases (MMP-9 and MMP-2) thereby also reducing pro-inflammatory TNF-α expression in a mouse model of traumatic SCI . The same group further demonstrated that melatonin’s protective role in SCI was related to the regulation of MAPK signaling pathways and the high-mobility group box 1 protein expression (HMGB1) in mice. They documented that melatonin treatment in SCI mice enhanced motor recovery, reduced the activation of p38 MAPK, JNK, and ERK1/2, and the expression of HMGB1 .
An earlier report also showed that activation of the endogenous melatonin system in the spinal cord reduced the generation, development, and maintenance of central sensitization, with a resultant inhibition of capsaicin-induced secondary mechanical allodynia and hyperalgesia . To this end, it was recently confirmed in an experimental model of SCI that exogenously administered melatonin reduced mechanical allodynia by altering the expression of water channel aquaporins .
Complementing the data regarding the neuroprotective effects in rodent SCI models, the beneficial neurobiological effects of melatonin have also been demonstrated in the rabbit SCI models. In these reports, both preischemic and postischemic administration of melatonin in a model of ischemia/reperfusion induced SCI following temporary aortic occlusion protected against free radical damage by induction of anti-oxidant enzyme levels and/or scavenging free radicals. This mitigated the need for extensive activation of endogenous anti-oxidant enzymes . Melatonin in combination with prophylactic zinc showed neuroprotection in a similar SCI model . In a recent study, it has been reaffirmed that melatonin application significantly abated the severity of SCI after temporary aortic occlusion in the rabbit . Further confirmation of neuroprotective efficacy of melatonin in traumatic SCI comes from a study conducted in rabbits wherein pinealectomy retarded the recovery rate after SCI, and administration of melatonin exogenously to the pinealectomized animals augmented the recovery .
The multifunctional efficacy of melatonin treatment after SCI has been shown in vivo by many investigations. We investigated the role of melatonin as an intervening agent for ameliorating Ca2+-mediated events, including activation of calpain in moderately severe experimental SCI . Calpain, a Ca2+-dependent neutral protease, is known to be a key player in the pathogenesis of SCI. In an acute SCI regimen, immunofluorescent labeling was used to identify calpain expression in neurons, glia, or macrophages. A combination of TUNEL and double immunofluorescent labelings was used to identify neuronal apoptosis in spinal cord. Furthermore, the effect of melatonin on axonal damage was assessed using an antibody, which was specific for dephosphorylated neurofilament protein. Treatment of SCI animals with melatonin attenuated calpain expression, inflammation, axonal damage, and neuronal death, indicating that melatonin was highly neuroprotective in this situation. Moreover, examination of levels of calpain and caspase-3 expression and activity indicated significant reductions in the proteolytic events in SCI animals after treatment with melatonin. Taken together, our studies strongly suggest that melatonin may be an effective neuroprotective agent for the treatment of SCI , and the results are summarized (Table 1).
Table 1. Efficacy of melatonin in diverse animal models of experimental SCI
|Type of SCI||Melatonin doses||Insights into melatonin efficacy in SCI|
|Studies in rats|
|Range of methods were used to induce SCI in rats, including compression, weight drop, and contusion. Both genders of Wistar and Srague-Dawley rats were used. Studies included both acute and chronic SCI spanning from days to weeks.||Range of melatonin doses were used from 10 to 250 mg/kg; applied as single bolus or multiple doses; delivered mostly immediately after SCI and in some studies continued from 1 to 10 days. Melatonin was also used in combination with methylprednisolone.||Melatonin was found superior to methylprednisolone but no cumulative advantage on the chronological recovery was seen with combination therapy; systemically applied pharmacological dose of melatonin boosted antioxidant defense systems after SCI; melatonin efficacy was found superior to other antioxidants in acute SCI; it was also found to be more effective than octreotide, an octapeptide that mimics natural somatostatin. Anti-inflammatory, anti-oxidative and anti-nitrosative action of melatonin were seen in SCI. Pinealectomy aggravated SCI, whereas exogenous melatonin had neurprotective effect in pinealectomized animals like that of injury-vehicle group. Melatonin reduced the expression and activity of matrix metallo-proteinases (MMP-9 and MMP-2) thereby reducing the TNF-α expression. Melatonin also reduced mechanical allodynia through regulation of aquaphorin-1 and rendered neuroprotection by attenuating proteolytic damage.|
|Studies in rabbits|
|Acute ischemic SCI induced in male New Zealand rabbits by clamping of thoraco-abdominal aorta at 1–2 sites for 20–30 min.||Melatonin doses were 10 mg/kg 10 min preischemia and postischemia or in combination with zinc sulfate.||Melatonin protected against acute SCI induced by ischemia/reperfusion; it showed significant neuroprotection in combination with prophylactic zinc and significantly reduced incidence of SCI after temporary aortic occlusion.|
|Studies in mice|
|Acute and chronic SCI induced by extradural aneurysm clip.||Combination of melatonin (dose) with dexamethasone.||Novel aspects of combination therapy as a significantly low dose of dexamethasone were effective.|
Contrary to the aforementioned beneficial reports in experimental SCI, melatonin failed to render any neuroprotection in a model of iatrogenic SCI for cases of elective spine surgery, wherein preoperative (20 min), intra-operative, or postoperative (1 h) doses (10 mg/kg) were applied to incomplete SCI rats. It is important to note that three other neuroprotective drugs tested, including methylprednisolone also did not improve motor fuction or spinal cord morphometry; hence, the authors have underscored the paramount significance of a total avoidance of primary injury altogether in elective spine surgery .
Traumatic brain injury
Clinicians and basic scientists are in agreement that a significant barrier in finding an effective therapy in traumatic CNS injuries lies in the enormous clinical heterogeneity in all forms of neurotrauma. Recently, a major step was taken in addressing this issue by a group of prominent organizations (National Institute of Neurological Disorders and Stroke, Brain Injury Association of America, Defense and Veterans Administration Brain Injury Center, and National Institute of Disability and Rehabilitation Research) that, together, outlined a new, multidimensional classification system for TBI. This is a major modification of the existing Glasgow Coma Scale and is expected to appropriately link specific patterns of brain and neurovascular injury with beneficial therapeutic interventions, thus, facilitating clinical trials . Over the years, there has been substantial evolution of the experimental models for TBI . A remarkable trend in progressive development of these models has been focused on: defining a temporal window that gives an oppurtunity for interventions, testing the safety and efficacy of delivery systems of agents and cells, and providing a better understanding of the cascades of gene expression and cell interactions both acutely and chronically after injury. The published literature on the clinical and experimental background may serve as great resource to appreciate the anticipated benefits of melatonin usage in clinics for TBI.
Melatonin efficacy in TBI
The neuroprotective efficacy of melatonin in TBI or craniocerebral trauma has been recently reviewed . Like other forms of neurotrauma, TBI involves multifactorial etiologies including free radical generation, which culminates in oxidative and nitrosative damage, disrupted macrocirculation and microcirculation in the vicinity of the injury, lymphocytopenia, opportunistic infections, perturbed sleep-wake cycles, suppression of nonspecific resistance, and toxicity caused by therapeutic agents. Together, these factors precipitate into the development of heterogenous clinical symptoms in the secondary phase. Melatonin has been adjudged as a protective agent against damage following TBI in several in vitro and in vivo studies . Melatonin as well as its metabolites are multi-faceted free radical scavengers and are effective against both oxygen and nitrogen-based reactants and may clearly attenuate neural damage resulting from craniocerebral trauma [73, 74]. Enhanced vulnerability of brain to such oxidative or nitrosative stress that escalates the damage following TBI may be handled more deftly by melatonin compared to other anti-oxidants. Infection and inflammation complicates TBI in surviving patients. Melatonin also aids in inhibition of pro-inflammatory cytokines and activation of adhesion molecules. It would consequently reduce lymphocytopenia and infections by opportunistic organisms. Moreover, the chronobiotic capacity of melatonin may also reset the natural circadian rhythm of sleep and wakefulness, which may serve as a major advantage of melatonin’s use as a therapeutic molecule after traumatic injury. It may also reduce the toxicity and enhance the efficacy of drugs used in clinical management of TBI such as steroidal or nonsteroidal anti-inflammatory agents, anti-ulcer agents, anti-psychotics/antidepressants, anti-epileptics and also anti-anemic drugs. Similar benefits of melatonin may be seen in other traumatic injuries to CNS as well. Finally, melatonin, not being restricted by the blood-brain barrier, reportedly reduces the contusion volume and stabilizes cellular membranes preventing vasospasm and apoptosis of endothelial cells that occurs as a result of TBI [32, 75].
Melatonin efficacy in ischemia/reperfusion
The integrity of the CNS is highly vulnerable to a transitory interruption of its blood supply; this is specially true of the brain which is susceptible to focal or global ischemia. Unless ischemia is promptly reversed, reperfusion produces further cerebral damage. Ischemia/reperfusion injury is a subset of CNS damage that is unfortunately common but also occurs with equal frequency in nonneural tissues. In the CNS, interruption of the blood supply followed by reperfusion may not always be a result of traumatic assault. However, as the damage is associated with free radical-mediated attack of essential molecules, it is included in this survey.
Several recent reviews [30, 31, 37] summarize the reports that have documented the neuroprotective effects of melatonin against ischemia/reperfusion injury to the brain. Ischemia (focal or global) causes extensive destruction of neural tissue and the primary objective in the clinic is to promptly reverse it. Procedures such as acute thrombolysis or defibrinogenation, however, are effective only in selective patients, and are associated with a significant risk of complications due to bleeding. To date, a number of neuroprotectants, which were initially found effective in experimental studies for stroke therapy, failed in clinical trials. At this juncture, it may be rational to test the efficacy of melatonin. The in vivo and in vitro evidence in favor of the use of melatonin to protect against focal and global cerebral ischemia/reperfusion injury has been reviewed . The benefits of conducting further experimental studies to examine any synergistic protective action by combining melatonin with thrombolysis, defibrinogenation or other neuroprotectants are emphasized. The planning of phase II and phase III trials in an attempt to define the potential benefits of melatonin as an acute stroke treatment in humans is suggested .
When the results of all the reports are considered, it is clear that endogenously-produced and exogenously-administered melatonin reduces the degree of tissue damage and limits the behavioral deficits associated with experimental models of stroke . This further suggests that melatonin efficacy should be seriously considered for clinical trial.
The most recent updates on melatonin’s efficacy in models of stroke come from another review where the potentially widespread applications of melatonin have been emphasized . These studies provide the preclinical tests for melatonin in acute stroke therapy, and its application at different treatment schedules. Advantageous characteristics of melatonin as a neuroprotective drug relates to its bioavailability, its potential for exerting wide-spread neuroprotective actions, further amplified and prolonged by its metabolites , its ability to reduce inflammation, its capability to protect cytoskeleton organization, and its anti-apoptotic actions. Moreover, melatonin does not interfere with the thrombolytic and neuroprotective actions of other drugs. An adequate safety profile of the drug has been underscored. The potential use of melatonin as a neuroprotective drug in clinical trials aimed at improving the acute focal or/and global cerebral ischemia has also been suggested in another review .