Exploring the exogenous and endogenous effects of melatonin on spinal cord injury

List of abbreviations

COX

cyclooxygenase

GSH

glutathione

IL-1α

interleukin-1α

IL-1β

interleukin-1β

iNOS

inducible nitric oxide synthase

L/D

light/dark

L/L

light/light

LPO

lipid peroxidation

MDA

malondialdehyde

MT

melatonin

NF-κB

nuclear factor-κB

NG-2

neuron/glial antigen 2

SCI

spinal cord injury

SOD

superoxide dismutase

TBI

traumatic brain injury

TNF-α

tumor necrosis factor-α

Introduction

Spinal cord injury (SCI) is a catastrophic incident with a global mortality rate ranging from 4.4% to 16.7%, often leading to neurological disability ( ). Traumatic injury to the spinal cord induces death in a number of local neurons and glia at the lesion site that cannot be recovered or regenerated. The mechanisms of secondary injury begin immediately after the primary insult and continue for weeks or months via a diverse array of pathophysiological processes, including inflammation, excitotoxicity, and oxidative cell damage ( ). These secondary insults lead to further destruction of neuronal and glial cells and to massive extension of the damage whereby the paralysis can extend to higher segments.

Disruption of circadian rhythm is a common feature in SCI individuals. Circadian misalignment can increase neuronal death, resulting in deterioration of sensorimotor functions and cognitive deficits ( ). In experimental studies, SCI was shown to lead to wide-ranging circadian rhythm disruption, including dysregulated rhythms of body temperature, and inflammatory gene expression ( ). Besides, circulating levels of serum melatonin (MT) has been altered in patients with cervical SCI ( ), suggesting alteration of diurnal rhythms negatively influencing the SCI recovery process.

As SCI results in permanent or long-term disability and poor quality of life, it imposes an enormous financial burden on society in terms of increased healthcare costs and decreased productivity ( ). Although efforts are currently being pursued to develop novel therapeutics to combat the pathogenesis of SCI, the effects of these interventions are generally limited. For example, exercise interventions, which are widely used to improve functional recovery following SCI, have an inadequate ability to improve motor function after SCI ( ).

MT is a neurohormone that is synthesized and released from the pineal gland in a 24-h diurnal pattern and exerts its actions in peripheral tissues. This pineal hormone is best known for maintaining the 24-h internal clock and has excellent antioxidant capacity ( ). MT exerts beneficial effects by altering the levels of oxidative stress markers, including malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and myeloperoxidase, which are generally reported to show abnormalities with progression of SCI ( ; ). MT has other biological functions in SCI, including reduction of pro-inflammatory molecules ( ) and regulation of autophagy ( ). Recently, we demonstrated that a significant reduction in endogenous MT levels disrupted neural remodeling, and homeostasis of endogenous MT levels contributed to excitatory synaptic formation and axonal outgrowth in a rodent model of SCI ( ), indicating MT replacement strategies improve the neuronal repair process following SCI.

In this chapter, we explore the promising molecular mechanisms of MT intervention that may be beneficial in the management of SCI. We discuss MT and exercise combination therapy in SCI. Finally, we also discuss the crucial role of endogenous MT, which may influence the recovery process after SCI.

Effects of exogenous melatonin on secondary injury after spinal cord injury

Neuronal death caused by primary injury from SCI cannot be prevented. Therefore, current research focuses on preventing the secondary injury cascade to mitigate progressive tissue damage, representing a novel strategy for the management of SCI pathology. The timeline of major pathological events of secondary injury and timeline-specific therapeutic strategies are shown in Fig. 1 . In this section, we will discuss recent findings on the effects of exogenous MT on SCI, particularly the neuroprotective mechanism of action of MT in secondary injury events.

Fig. 1, Timeline of the major pathological events after spinal cord injury (SCI) and the best therapeutic strategies for SCI. The secondary injury mechanisms are initiated after the primary insult and sustained over a long period. The events that occur after primary insults are divided into the acute (< 48 h), sub-acute (2 days–6 months), and chronic (> 6 months) phases. (A) In the acute phase, hemorrhage, ischemia, and edema cause cell dysfunction and death. In addition, the inflammatory cells infiltrate into the injured spinal cord, resulting in the release of pro-inflammatory cytokines. These excessive inflammatory mediators can disrupt the blood-spinal cord barrier, further exacerbating the injury. (B) In the sub-acute phase, glutamate excitotoxicity, excessive ROS generation, and the full-blown inflammatory response cause further cell death and damage to the spinal cord architecture. Importantly, glial scar formation initiates this phase. (C) In the chronic phases, axons continuously degenerate, and the glial scar matures, inhibiting the neuronal regeneration process. The inflammatory phenotype shifts from pro-inflammatory to anti-inflammatory in this phase. Cavity formation also appears, which suppresses axonal regrowth. During the acute and sub-acute phases of SCI, multiple pathological cascades trigger the cell death process, leading to greater damage than that caused by the primary insult. Therefore, the pathophysiological events associated with the acute and sub-acute phases are the ultimate targets for therapeutic interventions. ROS , reactive oxygen species; SCI , spinal cord injury.

Effects of exogenous melatonin on oxidative stress

Pre-clinical research in animal models has reported that increased production of reactive oxygen species (ROS) and the consequent oxidative stress are crucial pathological events in SCI, which lead to neurological deficits. Oxidative stress is a hallmark of the secondary injury in SCI. Much of the current literature on secondary injury after SCI has focused particularly on oxidative stress because down-regulation of its detrimental effects is considered a key strategy for therapeutic interventions ( ). After SCI, oxidative stress may increase the level of lipid peroxidation (LPO) ( ), as determined by quantifying the levels of MDA, GSH, and SOD. Electron resonance spectrometry was used to measure MDA levels and provided the first lines of evidence for ROS production in SCI. Seligman et al. reported that the level of MDA was significantly increased within the first 5 h following SCI. A recent study showed that the MDA level increased markedly after 1 day and peaked at 7 days after SCI ( ). SOD activity was decreased at 1 day following SCI ( ). Previous studies showed that MT and its derivatives scavenge free radicals and induce the activities of various antioxidant enzymes. Erşahin et al. investigated the impact of MT in a rat model of standard weight drop-induced moderate SCI and showed that MT significantly restored the MDA and GSH levels. Immediately after laminectomy, treatment of SCI rats with MT once daily for 10 days also resulted in restored GSH levels ( ). In addition, the pathophysiological mechanism was proposed to involve iron-catalyzed LPO contributing to autodestruction in the injured spinal cord regions ( ). Previous research established that ferrous iron can initiate brain LPO ( ). After SCI, the levels of free iron and MDA were significantly increased but markedly decreased by MT treatment ( ). investigated the effects of MT, prostaglandin E1, and oxytetracycline on LPO and antioxidant activities in an experimental model of SCI. The results showed that experimentally induced SCI reduced erythrocyte SOD and plasma GSH activities and increased tissue and blood MDA levels ( ). These physiological alterations were inhibited by MT, prostaglandin E1, and oxytetracycline treatments to varying degrees, with MT showing the greatest effect ( ). Taken together, pre-clinical trials suggest that MT may exert neuroprotective effects on SCI by reducing oxidative stress and promoting functional recovery after SCI ( Fig. 2 ).

Fig. 2, Schematic representation of the potential mechanisms of action of melatonin in spinal cord injury. Increased production of ROS, MDA, LPO, and consequent oxidative stress is a crucial pathological event in SCI. Melatonin can scavenge free radicals, increase the levels of antioxidant enzymes such as SOD and GSH, and reduce the levels of MDA and LPO following SCI. Moreover, inflammation can directly promote tissue damage and weaken functional recovery after SCI by generating a wide array of inflammatory molecules. However, exogenous melatonin can reduce the levels of inflammatory molecules, such as TNF-α and IL-1β, possibly by inhibiting NF-κB. Melatonin also reduces the expression of iNOS and COX. COX , cyclooxygenase; IL-1β , interleukin-1β; iNOS , nitric oxide synthase; LPO , lipid peroxidation; SOD , superoxide dismutase; GSH , glutathione; MDA , malondialdehyde; NF-κB , nuclear factor-κ light chain enhancer of activated B cells; ROS , reactive oxygen species; SCI , spinal cord injury; TNF-α , tumor necrosis factor-α.

Effects of exogenous melatonin on inflammation

Within a few hours following SCI, pro-inflammatory cytokines, such as interleukin (IL)-1α, tumor necrosis factor-α (TNF-α), and IL-1β, are activated ( ). After SCI, up-regulation of inducible nitric oxide synthase (iNOS) results in excessive production of nitric oxide (NO), which is abundant in astrocytes and microglial cells ( ). These excessive levels of NO further stimulate the synthesis of several pro-inflammatory cytokines ( ). These pro-inflammatory molecules are associated with activation of local microglia and astrocytes, which promote secondary injury after SCI. Several studies have postulated that MT may contribute to reduced inflammation after SCI. Previously, our group reported that iNOS mRNA expression was significantly lower in the MT treatment groups than SCI groups in the region of the injured spinal cord ( ). Moreover, a recent study showed that treatment with MT markedly inhibited the accumulation and proliferation of microglia and astrocytes in the injured spinal cord and suppressed TNF-α, IL-1β, and iNOS expression after SCI ( ). Those findings may explain the cellular and molecular mechanisms by which MT exerts its neuroprotective effects and contributes to functional recovery after SCI. In a mouse model of severe crush SCI, the inflammatory response appeared to be significantly attenuated at 14 days after MT treatment (10 mg/kg) ( ). Immunohistochemical analysis also showed that MT treatment markedly reduced IL-1β and neuron/glial antigen 2 levels ( ). The results of that study suggested that MT can decrease the expression and release of pro-inflammatory molecules, thereby inhibiting tissue damage from the secondary inflammatory response. Furthermore, TNF-α may play a pivotal role in the acute phase of SCI. conducted a study in radiation-induced SCI rats to assess the efficacy of MT on TNF-α expression and reported that TNF-α expression was markedly increased in the irradiated group compared with the normal group 3 weeks after injury. The most striking result was that the oral MT treatment group (100 mg/kg) showed significantly decreased TNF-α expression compared with the radiation group, suggesting that oral MT impedes the up-regulation of TNF-α expression after radiation-induced SCI ( ). Activation of the nuclear factor-κB (NF-κB) signaling has also been implicated in the induction of inflammation and is considered one of the pathophysiological causes of the spinal cord inflammatory response following SCI ( ). NF-κB further generates ROS, cyclooxygenase, and iNOS, which synergistically induce inflammation ( ). Several lines of evidence suggest that MT inhibits the expression of NF-κB and attenuates the production of pro-inflammatory cytokines ( ). Recent research on MT suggested that this pineal hormone reduces secondary injury severity and neuronal death after SCI ( ). Another study conclusively showed that MT treatment effectively decreased inflammation and tissue injury in experimental SCI ( ). Overall, MT may exert neuroprotective effects on SCI by reducing inflammation and tissue damage after SCI ( Fig. 2 ).

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