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.

(Reprinted from Yamazaki, K., Kawabori, M., Seki, T., & Houkin, K. (2020). Clinical trials of stem cell treatment for spinal cord injury. International Journal of Molecular Sciences , 21 , 3994. According to the Creative Commons License (CC-BY-NC-ND).)


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 ).


Effects of exogenous melatonin on autophagy


Autophagy plays a significant role in improving the neurological recovery process and enhances neurological function within a short time following acute SCI ( ). Surprisingly, inhibition of autophagy markedly increased apoptosis in experimental SCI rats ( ; ). In contrast, enhancing autophagy at the lesion site of the spinal cord significantly inhibited apoptosis ( ), suggesting that increased levels of autophagy can reduce apoptosis, contribute to the neurological recovery process, and improve neurological function after SCI. A systematic review and network meta-analysis reported that MT improved locomotor recovery by inhibiting neuronal cell apoptosis in a rat model of SCI ( ). Similarly, demonstrated that MT impeded neuronal cell apoptosis and encouraged locomotor recovery by activating the Wnt/β-catenin pathway following SCI. Previously, our group examined the endogenous and exogenous effects of MT on autophagic markers (beclin-1, LC3) in a contusion SCI rat model ( ). In that experiment, all groups were exposed to one of the following lighting conditions: 12/12 h light/dark (L/D) or 24/0 h constant light (LL). Western blot analysis showed that the transformation ratio of LC3-I to LC3-II protein was very low or undetectable in the normal control group ( ). Interestingly, the ratio was elevated after injury in a time-dependent manner, particularly in the SCI group under the L/D condition. At the site of the spinal cord lesion, the LC3-II protein level was increased at 3 days and peaked at 28 days post-SCI. In addition, no significant difference in beclin-1 expression was observed under the L/D condition, but its expression was significantly elevated under the LL condition. Surprisingly, exogenous MT treatment markedly decreased these effects under both L/D and LL conditions. Those findings suggest that autophagy-related cell death was increased in the spinal cord, and MT treatment accelerated motor recovery by down-regulation of LC3-II and beclin-1 ( ). These results corroborate with , who also reported that the beclin-1 protein level was significantly increased at the lesion site after SCI. However, as mentioned above, up-regulation of autophagy inhibited apoptosis in the spinal cord, inducing neurological repair following SCI. A recent study revealed that treatment with MT markedly increased the levels of autophagy-activated proteins, such as beclin-1 and LC3, but decreased the levels of apoptosis-activated proteins, such as caspase-3, caspase-9, and bax, in neurons after SCI ( ). Those findings indicate that MT can improve locomotor function by increasing autophagy as well as decreasing apoptosis after SCI in rats, probably via the PI3K/AKT/mTOR signaling pathway ( ). In summary, MT can increase autophagy, improve neuronal survival, and enhance locomotor function, while decreasing apoptosis in the spinal cord following SCI via the PI3K/AKT/mTOR signaling pathway. Alternatively, MT may improve neuronal survival by down-regulating autophagy-mediated apoptosis after SCI ( Fig. 3 ). Further studies are required to clarify the differential effects of MT on autophagy in several experimental models of SCI.




Fig. 3


Effects of exogenous melatonin on autophagy in the injured spinal cord after spinal cord injury. (A–C) Western blot analysis indicated that the autophagy-activating proteins (LC3-II and beclin-1) were decreased by melatonin treatment.

(Republished with permission from Park, S., Lee, S.-K., Park, K., Lee, Y., Hong, Y., Lee, S., Jeon, J.-C., Kim, J.-H., Lee, S.-R., Chang, K.-T., & Hong, Y. (2012). Beneficial effects of endogenous and exogenous melatonin on neural reconstruction and functional recovery in an animal model of spinal cord injury. Journal of Pineal Research , 52 , 107–119. Wiley. Permission conveyed through Copyright Clearance Center, Inc.)


Combination treatment strategy for spinal cord injury


Although the single-intervention strategy has been effective in pre-clinical trials, the recovery process remains ineffective, and targeting a single barrier has been unsuccessful for improving functional recovery in cases of SCI in clinical trials. In contrast, combination treatment strategies that address diverse aspects of the pathology of SCI have been developed to achieve functional recovery after SCI ( ; ). Most recent pre-clinical and clinical trials on functional recovery after SCI have focused on several combination treatment strategies, including (i) antioxidants plus exercise and (ii) glucocorticoids plus antioxidants ( ). In this section, we discuss recent studies focusing on the beneficial effects of MT plus exercise on SCI.


Melatonin combined with exercise


Based on recent studies of SCI, the combination of MT and exercise was hypothesized to show synergistic effects, accelerating motor function recovery. As indicated previously, MT reduces LPO and increases the levels of several antioxidant enzymes after SCI ( ; ; ), indicating that MT itself is a super antioxidant. On the other hand, exercise interventions can influence structural and functional changes in the cerebral cortex, spinal cord, and skeletal muscle, thereby promoting neural and muscular function after SCI. Exercise interventions can accelerate nerve regeneration with functional recovery ( ; ) and sustain the functionality of spinal cord neurons ( ). Exercise can enhance neurological function following SCI but inadequate to improve motor function ( ). These limitations may be due to insufficient neurotrophic factor production by exercise training. In addition to exercise, therefore, several combination strategies have been investigated. Many studies have shown that antioxidants, such as MT, combined with exercise can considerably improve functional recovery following SCI and alleviate spasticity in rats compared with single interventions alone ( Fig. 4 ). The benefit of this combination strategy is that MT can diminish the side-effects related to exercise, such as fatigue and impairment ( ). Recently, we reported synergistic effects of MT and exercise on neuronal repair and functional recovery in a contusion-induced SCI animal model ( ), suggesting excessive production of iNOS accelerates secondary damage to spinal tissue, which may be reversed by MT combined with exercise. The results of that study showed that MT combined with exercise markedly elevated hind limb movement, decreased iNOS expression, and increased the proportion of motor neurons in the ventral horn compared with exercise intervention alone ( ). Previously, we also reported synergistic effects of MT and exercise in a rodent SCI model, with notable behavioral enhancement, histological recovery, and elevated numbers of BrdU/nestin double-positive endogenous neural stem/progenitor cells ( ). This study further demonstrated that MT and exercise intervention had a synergistic effect on nestin expression in the rostral perilesion site, suggesting that nestin may be associated with functional recovery. Using Golgi-Cox analysis, we showed that MT combined with exercise elevated dendritic spine density ( ). Previously, our group also comprehensively reviewed the therapeutic effects of MT combined with exercise on SCI. In this review, we suggest that MT combined with exercise would not only enhance functional recovery but also alleviate the secondary tissue damage after SCI ( ). Finally, we propose that future studies should explore the effects of MT and other antioxidants in combination with exercise on SCI recovery, representing a novel strategy for faster recovery after SCI ( ). Finally, the combination of MT and exercise decreases the secondary injury related to SCI, and thus MT can reduce the side-effects associated with exercise.


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Nov 9, 2024 | Posted by in NEUROLOGY | Comments Off on Exploring the exogenous and endogenous effects of melatonin on spinal cord injury

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