Pharmacological Effects of Melatonin as Neuroprotectant in Rodent Model: A Review on the Current Biological Evidence

Abstract

The progressive loss of structure and functions of neurons, including neuronal death, is one of the main factors leading to poor quality of life. Promotion of functional recovery of neuron after injury is a great challenge in neuroregenerative studies. Melatonin, a hormone is secreted by pineal gland and has antioxidative, anti-inflammatory, and anti-apoptotic properties. Besides that, melatonin has high cell permeability and is able to cross the blood–brain barrier. Apart from that, there are no reported side effects associated with long-term usage of melatonin at both physiological and pharmacological doses. Thus, in this review article, we summarize the pharmacological effects of melatonin as neuroprotectant in central nervous system injury, ischemic-reperfusion injury, optic nerve injury, peripheral nerve injury, neurotmesis, axonotmesis, scar formation, cell degeneration, and apoptosis in rodent models.

Introduction

Melatonin (5-methoxy-N-acetyltryptamine) is a derivative of tryptophan produced by pineal gland in response to signals received from the postganglionic fibers. Production of melatonin in the body is controlled by the circadian rhythm. Blood concentration of melatonin fluctuates according to the surrounding light intensity where dark conditions increases melatonin level in blood and decreases in light (Gooley et al. 2011; Hardeland 2013) as melatonin release is controlled by the diurnal cycle. Daylight stimulus activates melanopsin breakdown in retinal photoreceptive ganglion cells via the retinohypothalamic pathway and stimulates γ-aminobutyric acid (GABA)-ergic suprachiasmatic nucleus (SCN) projections towards paraventricular nucleus (PVN) of the hypothalamus. This subsequently causes preganglionic symphathetic neurons in the intermediolateral column of the spinal cord (IML) to supress melatonin production and results in inhibition of melatonin synthesis (Hattar et al. 2002; Kalsbleek et al. 1999). When absent of light stimulus, GABA-ergic activity is arrested in addition with the ongoing active glutamatergic inputs allowing re-autonomic PVN to reactivate and further release neurotransmitter norepinephrine form the superior cervical ganglion (SCG) (Drijfhout et al. 1996; Hermes et al. 1996; Perreau-Lenz et al. 2004). Norepinephrine (NE) released from the nerve terminals of SCG in the pineal acts primarily on β-adrenergic receptors in the pinealocyte membrane which increases the intracellular cAMP (Kebabian et al. 1975). Hence, this results in increased activity of the rate-limiting enzyme of melatonin synthesis serotonin-N-acetyltrasnferase (NAT) (Drijfhout et al. 1996). This is a rate-limiting step in melatonin synthesis as the NAT activity is extremely sensitive to changes in light (Coon et al. 1995). Activity of NAT can rapidly change depending on exposure of light. NAT activity increases 10- to 100-fold during night time as compared to day time (Borjigin et al. 1995; Roseboom et al. 1996). The next step of melatonin synthesis involves the transfer of methyl group to N-acetylserotonin by hydroxyindole-O-methyl transferase (HIOMT) to yield melatonin (Weissbach et al. 1960) (Fig. 1).

Fig. 1.

The mechanism of melatonin production in pineal gland

There are two subtypes of melatonin receptors identified in mammals, namely MT1 and MT2. They are members of the seven transmembrane G protein-coupled receptors superfamily (Witt-Enderby et al. 2003). MT1 and MT2 are the main G protein-coupled melatonin receptors that are widely found in human central nervous system (CNS) including cerebellar cortex, hippocampus, thalamus and retina (Ng et al. 2017; Savaskan et al. 2002, 2005; Witt-Enderby et al. 2003). The physiological function of melatonin is exerted through binding to MT1 and MT2 receptors, which then leads to activation of different cellular responses (Ekmekcioglu 2006; Uz et al. 2005; Witt-Enderby et al. 2003). MT1 receptor is a pertussis toxin-sensitive guanine nucleotide-binding protein that mediates inhibition of adenylyl cyclase in local tissues (Carlson et al. 1989). On the other hand, MT2 receptor is a G protein-coupled receptor and is involved in inhibiting adenylyl cyclase and soluble guanylyl cyclase pathways (Petit et al. 1999). Both melatonin receptors MT1 and MT2 have shown to activate phospholipase C pathway, which then leads to an increase in levels of inositol triphosphate (IP3) and 1,2-diacylglycerol (DAG), which are linked to the pertussis toxin-insensitive Gq protein (Jockers et al. 2008; Sharkey and Olcese 2007; Witt-Enderby et al. 2003). Interestingly, MT1/MT2 heterodimer has triggered a greater activation of these pathways, suggesting that there is an allosteric interaction between the two receptors (Jockers et al. 2008; Sharkey and Olcese 2007; Witt-Enderby et al. 2003). Activation of the Gi protein inhibits AC/cAMP/PKA/CREB pathway where the MT1 and MT2 receptors are found tightly coupled to the pertussis toxin-sensitive Gi protein (Rivera-Bermúdez et al. 2004; Tosini et al. 2014). Besides that, GC/cGMP/PKG pathway is inhibited through MT2 receptor binding via Gi activation (Petit et al. 1999).

Melatonin has pleiotropic effects and targets both cells and mechanisms. Apart from that, melatonin can enter body fluid, cell or cell compartment easily because of its amphiphilicity (Hardeland and Pandi-Perumal 2005). The antioxidant effects of melatonin includes direct radical scavenging, enzymatic regulation of oxidant formation, and mitochondrial radical avoidance (Dubocovich et al. 2010). Specificity, melatonin upregulates antioxidant enzymes such as glutathione peroxidase (Gómez et al. 2005), glutathione reductase (Liu and Ng 2000), Cu, Zn- and/or Mn-superoxide dismutases (Rodriguez et al. 2004), catalase (Tomás-Zapico et al. 2002) and downregulates prooxidant enzymes such as 5- and 12-lipoxygenases (Manev et al. 1998; Uz and Manev 1998; Zhang et al. 1999), nitric oxide (NO) synthases (Bettahi et al. 1996; Reiter et al. 1998; Rogério et al. 2002) production. Moreover, melatonin increases electron flux, proton potential, ATP synthesis, in turn results in preventing electron leakage and enhancing complex I and complex IV activities (Martín et al. 2002; Acuña-Castroviejo et al. 2003). Melatonin also decreases 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Ca²⁺ release which leads to decrease cytochrome C generation and caspase 3 activation (Petrosillo et al. 2009; Waseem et al. 2016). Direct antioxidant effects of melatonin includes interactions of melatonin with oxidizing free radicals such as carbonate radicals (Hardeland et al. 2003), singlet oxygen (Matuszak et al. 2003), ozone (Hardeland 1997), and protoporphyrinyl and substituted anthranilyl radicals (Chandra et al. 2000). The mechanism of scavenger cascade with a possible sequence of melatonin → cyclic 3-hydroxymelatonin → N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) → N-acetyl-5-methoxykynuramine (AMK), where melatonin can be alternately converted to AFMK directly (Tan et al. 2003). From melatonin to AFMK, up to four free radicals can be consumed (Tan et al. 2003). Moreover, melatonin and its metabolites, 6-hydroxy, AFMK, AMK and cyclic 3-hydroxy melatonin also inhibit expression of inflammatory cytokines (interleukin-6, interleukin-8 and tumor necrosis factor-α (Baykal et al. 2000), matrix metalloproteinases (MMP-9 and MMP-2) (Esposito et al. 2008; Ebadi et al. 2005), cyclooxygenase (COX), prostanoids, NO, neuronal (nNOS), and inducible nitric oxide synthase (iNOS) (Acuña-Castroviejo et al. 2005; Deng et al. 2006; León et al. 2006; León et al. 2000; Tapias et al. 2009) and reverse the inflammatory response (Bilici et al. 2002; Mayo et al. 2005a, b; Perianayagam et al. 2005; Tarocco et al. 2019). On the other hand, melatonin can exert anti-apoptotic actions through inhibition of cytochrome c release (Wang et al. 2009), reducing caspase-1 and caspase-3 activation, increasing anti-apoptotic proteins (Bcl-2, Bcl-xL) expression (Ling et al. 1999), decreasing pro-apoptotic proteins (Bad, Bax) expression (Koh 2008). All these events inhibit mitochondrial permeability transition pore opening (Andrabi et al. 2004) and prevent apoptosis (Sun et al. 2002; Alvira et al. 2006). Thus, this review summarizes and discusses the potential therapeutic application of melatonin in neuroptotection based on current biological evidence.

Effects of Melatonin on Central Nervous System

Neuronal cell membranes consist of high levels of lipids, which makes it more prone to oxidative damage as lipids are easily oxidized (Hulbert et al. 2007). Moreover, oxidative stress also impairs pineal gland SCN function and affects melatonin production and circadian rhythms (Wu et al. 2003; Wu and Swaab 2005). The decline in melatonin production has been associated with neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Reiter et al. 1994; Reiter 1995a, b; Adi et al. 2010).

AD is the most common form of dementia that is characterized by progressive decline in learning and memory. In addition to degeneration and loss of neurons, the major pathological hallmarks of AD are amyloid-β plaques deposition and intracellular neurofibrillary tangles (NFTs), mainly containing hyperphosphorylated microtubule-associated protein tau formation in the brain (Billingsley and Kincaid 1997; Braak et al. 1994; Brion et al. 2001).

Several reviews have summarized the neuroprotective effects of melatonin against Alzheimer’s disease (Alghamdi 2018; Cardinali et al. 2012; Götz et al. 2018; Lin et al. 2013; Shukla et al. 2017). Post-mortem AD brains has shown markers of increased free-radical production, lipid peroxidation, oxidative DNA, and protein damage compared to age-matched healthy controls, and these are factors likely result in neuronal cell death (Lyras et al. 1997; Mecocci et al. 1994; Mullaart et al. 1990; Wang et al. 2005a, b, c). Melatonin supplementation has shown to improve sleep and slow down the progression of cognitive impairment in AD patients through antioxidative property (Brusco et al. 2000; Cardinali et al. 2010). Various studies have demonstrated that melatonin inhibits Aβ generation and arrests the formation of amyloid fibrils by a structure-dependent interaction with Aβ (Matsubara et al. 2003). Aβ generation is regulated by glycogen synthase kinase-3 (GSK-3), melatonin inhibits GSK-3 activation and upregulates of c-Jun N-terminal kinase (JNK) production resulting in elevated matrix metalloprotease activity and increased degradation of Aβ (Donnelly et al. 2008). Furthermore, melatonin inhibits caspase-3 activation that in turn decreases β-secretase activation and downregulates Aβ production through Bcl-2 expression in AD transgenic mice (Ling et al. 1999; Tesco et al. 2007). Melatonin prevents apoptosis via regulating Bcl-2/Bax, glutathione, and its enzymes and increases activation of prosurvival PI3K/Akt pathway (Shukla et al. 2017). Apart from that, melatonin treatment reversed mitochondrial dysfunction in APP/PS1 transgenic mice. This was accompanied by observations of decreased Aβ levels by two- to fourfold in cortex, hippocampus, and striatum brain region. This is possibly mediated through restoration of mitochondrial respiratory rates, membrane potential, and ATP levels. However, melatonin receptor antagonists block the restorative effect of melatonin on mitochondrial indicating melatonin receptor signaling, especially via MT2 receptor, is required for the full effect (Dragicevic et al. 2011).

Inhibition of tau hyperphosphorylation has been demonstrated to be one of the therapeutic targets of AD treatment. Studies have demonstrated that melatonin efficiently attenuates Alzheimer-like tau hyperphosphorylation via inhibition of protein kinases (glycogen synthase kinase-3, isoproterenol-induced protein kinase A) and activation of phosphatases (phosphatase-2A) in wortmannin, isoproterenol, calyculin A treated rats (Liu and Wang 2002; Wang et al. 2005a, b, c; Yang et al. 2011). In animal studies, administration of melatonin improves behavioral impairments of light-exposed rats through reduction in expression of endoplasmic reticulum (ER) stress-related proteins such as immunoglobulin-binding protein (BiP)/glucose-regulated protein, 78 kDa (GRP78) and C/EBP homologous protein (CHOP)/growth arrest and DNA damage 153 (GADD153), increase in number of rough ER, free ribosome that in turn decreases oxidative damage in mitochondria (Ling et al. 2009). Accumulated data provided evidence that melatonin inhibits protein kinase A (PKA) activity through melatonin receptor coupled inhibition of adenylyl cyclase and reduction of cyclic adenosine monophosphate (cAMP) (Peschke et al. 2002). Melatonin inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inflammation pathway that leads to reduction in neuritic injury, hyperphosphorylation of the tau protein neurofibrillary tangles formation (Hardy and Higgins 1992; Chuang et al. 1996).

Several reviews have summarized the neuroprotective effects of melatonin in PD (Mack et al. 2016; Singhal et al. 2012; Srinivasan et al. 2011). PD is the second most prevalent neurodegenerative disease after AD. PD is characterized by loss of dopaminergic neurons, presence of Lewy bodies and dysregulation of ubiquitin proteasomal system, mitochondrial metabolism, permeability and integrity, and cellular apoptosis resulting in bradykinesia, postural instability, rigidity and tremors (Tansey et al. 2007; Maguire-Zeiss and Federoff 2010). A 24-h study on melatonin rhythm found that melatonin level was reduced in PD patients (Videnovic et al. 2014). Moreover, decreased expression of MT1 and MT2 receptors was reported in post-amygdala and substantia nigra regions in PD patients (Adi et al. 2010). It was double confirmed by MT1/MT2 double knockout mice and standard behavioral test for spatial/reference learning (Barnes Maze) and basic working memory (Y-Maze) were used to test cognitive performance. Results revealed enhanced cognition in the MT1/MT2 double knockout mice as compare with receptor-intact control mice (O’Neal-Moffitt et al. 2014). Apart from that, using Luzindole (MT2 receptor antagonist) proved that melatonin decreased synaptic efficiency and/or excitability of hippocampal neurons via MT2 receptor (Hogan et al. 2001, Wang et al. 2005a, b, c). However, other studies observed hippocampus long-term potentiation (LTP) inhibition by melatonin that was blocked by MT receptors antagonist and by selective MT2 receptor antagonist, suggesting a mediated effect by MT2 receptor (Hogan et al. 2001). This hypothesis was confirmed using MT1 knockout and MT2 knockout mice, in which the inhibitory LTP action of melatonin was lost in MT2^−/− mice, but not in MT1^−/− mice (Videnovic et al. 2014).

Various drugs including reserpine, methamphetamine, 6-hydroxydopamine (6-OHDA), MPTP have been used to induce PD animal model (Bezard et al. 1998; Carlsson et al. 1957; Carlsson 1959; Dabbeni-Sala et al. 2001; Joo et al. 1998a, b; Ilijic et al. 2011; Przedborski et al. 2001). These drugs induce free-radical production and regulate mitochondrial electron transport chain and induce apoptosis of the dopaminergic neurons in substantia nigra (Nicklas et al. 1985; Mizuno et al. 1987; Srinivasan et al. 2005; León et al. 2006). It has been proven that melatonin prevents lipid peroxidation, restores tyrosine hydroxylase (TH) activity, elevates dopamine content, and dopamine-1 receptor in 6-OHDA induced PD rats (Joo et al. 1998a, b). Apart from that, melatonin inhibits dopaminergic neurons degeneration through attenuation of nitrosative and oxidative stress, intracellular calcium ion and phosphorylated p38 mitogen-activated protein kinases (MAPK), NFκB-induced inflammatory response in MPTP-induced PD rats (Ma et al. 2009; Niranjan et al. 2010). Evidence found that melatonin reverses methamphetamine/amphetamine-induced brain damage and functional decline by increasing striatal dopamine and dopamine transporter binding sites, regeneration of nerve terminals, restoring expressions of tyrosine hydroxylase (TH), synaptophysin, and growth-associated protein-43, preserving the levels of vesicular monoamine transporter 2 (VMAT-2) and phosphorylated TH (Itzhak et al. 1998; Kaewsuk et al. 2009; Mukda et al. 2011). Melatonin prevents rotenone-induced brain damage by preventing nigrostriatal degeneration, alpha-synuclein aggregation, hydroxyl radical generation, glutathione reduction, and changes in the catalytic activities of superoxide and catalase in substantia nigra (Saravanan et al. 2007; Lin et al. 2008).

In conclusion, the protective roles of melatonin in PD include free-radical scavenging (Reiter et al. 2001), mitochondrial modulating (Idowu et al. 2017), growth factor promoting (fibroblast growth factor 9), which can upregulate heme oxygenase-1 and gamma-glutamylcysteine synthetase expressions (Huang et al. 2009), anti-apoptotic molecule (Srinivasan et al. 2005). However, animal models developed so far may not correctly represent the clinical features of PD. Hence more studies, ideally using clinical subjects, are required to prove the benefit of melatonin in PD.

Effects of Melatonin on Ischemic-Reperfusion Injury

Several reviews summarize the neuroprotective effects of melatonin against ischemic-reperfusion injury to the brain (Cheung 2003; Cervantes et al. 2008; Alonso-Alconada et al. 2013; Naseem and Parvez 2014; Samantaray et al. 2009; Singhanat et al. 2018). Ischemic-reperfusion injury occurs commonly after a cerebrovascular accident and lead to long-term disability or death (Kawachi et al. 1993; MacMahon and Rodgers 1994; Caplan et al. 2017). Various clinical conditions such as conduction failure, endoneurial edema, multifocal, or diffuse loss of nerve fibers, axonal degeneration, and segmental demyelination are associated with ischemic-reperfusion injury (Nukada et al. 1993). Extensive damage is normally halted by restoring blood supply to the ischemic tissue as soon as possible since after initiation of symptoms. However, it is known that reperfusion after ischemic insult may increase the amount of reactive oxygen species that causes damage to the cellular components (Grace 1994). Following cerebral asphyxia, hypoxic-ischemia starts a multi-faceted cascade of events that ultimately causes cell death and often damages the whole brain (Hilario et al. 2006). It all begins when the reduction in oxygen and blood supply induces a decrease in oxidative phosphorylation and the brain converts to anaerobic metabolism to sustain functional ability. Anaerobic metabolism leads to rapid depletion of ATP, accumulation of lactic acid, and failure of ion pumps, resulting in a massive entry of sodium, calcium, and water into the cells that leads to production of reactive oxygen species, increase in free cytosolic calcium concentrations and the decrease in mitochondrial function ultimatley resulting in cell death.

Melatonin possesses neuroprotective effects against ischemic-reperfusion injury. Sayan et al. reported that after an ischemic-reperfusion injury, melatonin treatment resulted in increase in superoxide dismutase enzyme activity in sciatic nerve preventing neuropathological changes (Sayan et al. 2004). Sinha et al. demonstrated that 20 mg/kg and 40 mg/kg of melatonin decreased the production of malondialdehyde and glutathione levels in middle cerebral artery occlusion rats (Sinha et al. 2001). The evidence suggests that melatonin may confer neuroprotection against focal ischemia because of its antioxidant property.

Similarly, Guerrero et al. reported that melatonin protected the neuronal structures against transient ischemic insult by inhibiting NO and cyclic guanosine monophosphate (cGMP) production in the brain of Mongolian gerbil (Guerrero et al. 1997). NO is a free radical that significantly contributes to cell damage in ischemic-reperfusion injury (Guerrero et al. 1997; Iadecola 1997). Levels of nitrite, nitrate, and cGMP are known to increase during ischemic-reperfusion injury and thus are good indicators of NO production (Guerrero et al. 1997). Another study has proven that melatonin protected mitochondria in fetal rat brain against ischemic-reperfusion-induced damage (Nagai et al. 2008). Melatonin might act by inhibiting NO synthase, the enzyme that synthesizes NO.

Cumulative evidence showed that pretreatment with melatonin inhibited peroxynitrite formation, dysregulated mitochondrial HtrA2-PED signaling, and optimized survival rate of endothelial cells in an ischemic-like injury oxygen–glucose deprivation (OGD) model (Zou et al. 2002; Han et al. 2011; Tao et al. 2012). The OGD method induced peroxynitrite formation, causing disruption of the mitochondrial membrane and consequently releasing HtrA2 into the cytoplasm and inducing caspase-dependent apoptosis via binding and degradation of apoptosis protein inhibitors, resulting in pro-apoptotic effect induced by nitrosative stress (Suzuki et al. 2004; Han et al. 2011). Hence, the effect of melatonin on peroxynitrite inhibition indicates potential use of melatonin in stroke patients.

Besides that, various others studies showed that melatonin treatment also decreased blood–brain barrier permeability and reduced hemorrhagic risk of tissue plasminogen activator in brain ischemic-reperfusion model (Manev et al. 1996; Pei et al. 2003; Chen et al. 2006; Kaur et al. 2008; Babaei-Balderlou et al. 2010; Alluri et al. 2016). Previous studies reported that hyperpermeability of the blood–brain barrier mediated by interleukin-1β (IL-1β) and MMP-9 among others is one of the causes of traumatic brain injury, ischemia, and stroke (Didier et al. 2003; Simi et al. 2007; Alluri et al. 2016). Melatonin enhances the integrity of blood–brain barrier by reducing IL-β and inhibiting MMP-9 production (Alluri et al. 2016). Moreover, melatonin reduces the risk of intracerebral hemorrhage, which is known to be caused by the use of recombinant human tissue plasminogen activator for treating acute ischemic stroke (Wardlaw et al. 2012). Thus, it is suggested that the combinational treatment with melatonin and tissue plasminogen activator may enhance the efficacy of plasminogen activator. Additionally, melatonin was able to reduce edema, thus possibly preventing the damage caused by ischemic-reperfusion towards microvasculature. Melatonin was also found to reduce infiltration of inflammatory cells, leukocytes and microglia (Liang et al. 2014; Xu et al. 2017). Moreover, melatonin reduced the expression of the zonula occludens-1 (ZO-1) and water channels aquaporins-4 (AQP-4). Both proteins are important in maintenance of influx and efflux of transmembrane water and structure of the mucosal and vascular endothelial barrier in the brain, thus protecting against edema (Gunzel and Yu 2013; Liu and Agre 2013; Xu et al. 2017).

A few studies have demonstrated that pinealectomized and ischemic rats with bilateral carotid artery occlusion showed a decreased number of hippocampal pyramidal neuron population in Cornu Ammonis (CA)1 and CA4 regions of the forebrain. However, the reduction of neuron was not seen in control rats (Cuzzocrea et al. 2000; De Butte et al. 2002). These studies suggest that melatonin might be crucial in protecting these neuron populations in the event of cerebral ischemia. Cho et al. experimented using forebrain ischemic model where melatonin was administered intraperitoneally after reperfusion at 2 and 6 h in albino Wistar rats. Their results showed a significant increase in CA1 neuronal density. On the other hand, delayed melatonin treatment at 1 h after reperfusion or 30 min before reperfusion onset was ineffective in ischemic protection of CA1, suggesting that melatonin is most effective if administered early in ischemic-reperfusion event (Cho et al. 1997). The exact mechanism for the protective effect of melatonin is unknown, but it might be attributed to its antioxidant properties (Cho et al. 1997).

Various studies showed that melatonin was able to reduce the volume of infarct following transient focal cerebral ischemia (Sinha et al. 2001; Pei et al. 2002; Pei et al. 2003; Lee et al. 2004). Delayed treatment of melatonin at the onset of reperfusion reduced the size of both striatal and cortical infarction, and this is favorable in improving neurobehavioral and electrophysiological outcomes (Lee et al. 2004). Studies performed on pinealectomized rats demonstrated that treatment with melatonin before ischemic reperfusion was able to prevent the worsening of brain injury and reduced infarct volume where administration of melatonin with dose of 4 mg/kg reduced the infarct volume by 40% (Manev et al. 1996; Joo et al. 1998a, b; Kilic et al. 1999). Consistent with the findings from previous research, Manev et al. demonstrated that melatonin deficiency led to increased brain vulnerability in a focal brain ischemia model (Manev et al. 1996). The pinealectomized rats with cerebral artery occlusion had shown greater exaggeration of infarct volume compared to the rats with the intact pineal gland of the same condition (Manev et al. 1996).

A recent study done by Wei et al. showed that melatonin treatment reduced the infarct area and death of neurons caused by reperfusion stress by activating optic atrophy 1 (OPA1)-related mitochondrial fusion via the Yap-Hippo pathway. It was found that during ischemic-reperfusion injury, Yap-Hippo signaling was halted, thus reducing the OPA1-related mitochondrial fusion; and melatonin was found to reverse such effect (Wei et al. 2019). OPA1 plays a significant role in mitochondrial DNA exchange and recovery and can inhibit reperfusion injury in the brain (Zhang et al. 2016). Lately, the potential use of melatonin as a neuroprotective drug for patients suffering from global or acute focal cerebral ischemia has been investigated through clinical trials (Cheung 2003; Reiter et al. 2005). Treatment of spinal cord injury animals with melatonin attenuated calpain expression, inflammation, axonal damage, and neuronal death, indicating that melatonin was highly neuroprotective in this situation.

The role of melatonin receptors in ischemia/reperfusion injury, research found that the infarct volume and brain edema are same in MT1 and MT2 knockout (mt1/2)/) and wild-type (WT) animals. The neuroprotective effect of melatonin in mt1/2)/) animals better than in WT animals. Melatonin reduced CREB, ATF-1, and p38 phosphorylation in both mt1/2)/) and WT mice, while p21 and JNK1/2 proteins expression were decreased only in melatonin-treated WT animals. In conclusion, the neuroprotective effects of melatonin through membrane receptors independent pathway (Kilic et al. 2012). Other studies indicated that using MT2 receptor antagonist (luzindole), melatonin elicits its neuroprotective effect in ischemic stroke through MT2 receptor (Chen et al. 2015, Lee et al. 2010).

Effects of Melatonin on Optic Nerve Injury

Optic nerve degeneration is often caused by transection of the optic nerve near the posterior pole of the eye (Kilic et al. 2002). Transection of the optical nerve axon close to the cell body can cause loss of retinal ganglion cells (RGCs) (Garcia-Valenzulela et al. 1994). RGCs can undergo retrograde degeneration resulting in apoptosis, which involves the activation of caspase-3 and Bax (Kermer et al. 1998).

A recent study revealed that melatonin inhibited apoptosis of RGCs in traumatic optic neuropathy (TON). Wei et al. demonstrated that during TON, an increased number of caspase-3 positive cells and terminal-deoxyucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells were noted, suggesting the apoptotic events in the RGCs (Wei et al. 2017). After melatonin treatment, they found that the number of caspase-3 positive cells and TUNEL-positive cells had decreased, thus indicating the role of melatonin in inhibiting apoptosis (Wei et al. 2017).

Aranda et al. reported that melatonin preserved anterograde transport of cholera toxin β-subunit (CTB) from retina to superior colliculus (SC). This study was conducted on optic neuritis (ON) rat model that was induced through single microinjection of bacterial lipopolysaccharide (LPS) directly into the optic nerve. Results showed that melatonin treatment preserved CTB labeling in the SC whereas, without melatonin treatment, CTB staining was less intense in the whole retinotopic projection towards the SC (Aranda et al. 2016). Hence, this suggests that melatonin could preserve anterograde transport of CTB from the retina to the SC. Moreover, melatonin was found to prevent astrocytosis, microglial reactivity, demyelination, and loss of axon and RGC induced by LPS. Furthermore, melatonin prevented the reduction of pupillary light reflex, visual evoked potentials, partly lipid peroxidation that was caused by the experimental ON as well as prevented the increase in NO synthase 2, tumor necrosis factor (TNF)-α, and cyclooxygenase-2 levels (Aranda et al. 2016).

A few previous studies have investigated the protective effects of melatonin on eye pathologies such as ischemic-reperfusion-associated retinal edema uveitis. Retrograde degeneration of the optical nerve was induced by axotomy where the optical nerve was cut near the posterior pole of the eye positioned inside the orbital cavity. Their results demonstrated that the injury was aggravated in pinealectomized mice, but the effect could be reversed with exogenous administration of melatonin. However, there was no change in axotomized RGCs of the non-pinealectomized animals after exogenous administration of melatonin (Kilic et al. 2002). This suggests that melatonin plays a crucial role in recovery and therefore, pharmacological prescription of melatonin may be useful for an individual who experiences optic nerve injury.

Tang et al. showed that melatonin therapy could increase the survival rate of RGCs in ischemic-reperfusion injury-inflicted rats (Tang et al. 2006). This is possibly mediated through suppression of NO synthesis which thickens the inner retinal layer thickness and causes nuclear and morphological changes of the retina (Siu et al. 2004; Tang et al. 2006). When tissue is under hypoxic conditions, the heterodimer hypoxia-inducible factor (HIF)-1α and HIF-1β transcribes several genes associated with cellular adaptation to hypoxia such as NO and vascular endothelial growth factor (Xiao et al. 2013). Excessive expression of HIF-1α can eventually lead to cell death (Weidemann and Johnson 2008). Park et al. reported that melatonin treatment was able to inhibit the expression of HIF-1α, possibly mediated through the antioxidant properties of melatonin (Park et al. 2012). This resulted in reduced hypoxic response, therefore increasing the survival rate of RGCs in the ischemic retina (Park et al. 2012).

Effects of Melatonin on Peripheral Nerve Injury

Several reviews documented the neuroprotective effects of melatonin against PNI (Benga et al. 2017; Mekaj et al. 2014; Odaci and Kaplan 2009; Uyanikgil et al. 2017). PNI can be caused by traumatic or iatrogenic events, resulting in demyelination, axonal degeneration, or both (Antoniadis et al. 2014). PNI causes loss of sensory, autonomic, or motor functions in the denervated body segments and generally leads to acute functional deficits (Navarro 2015).

Turgut et al. (2005a) investigated the effects of melatonin on production of collagen and formation of neuroma after PNI and found that melatonin improved axonal regeneration and inhibited neuroma formation. Administration of exogenous melatonin inhibited the formation of collagen in neuroma of a suture repair site, thus promoting nerve regeneration (Turgut et al. 2005b). In pinealectomized rats, increasing Type I and Type III collagen production prevented sprouting of axons reaching end-organs and affecting nerves repair (Turgut et al. 2005b). Moreover, increased number of axons, increased thickness of the myelin sheath, and cross-sectional area of the axon were also found in pinealectomized rats (Turgut et al. 2006a, b). The results suggest that melatonin affects morphologic features of the peripheral nerve tissue and hence, deficiency in melatonin might contribute to degenerative diseases associated with peripheral nerves. Deficits of melatonin/MT2 signaling affect cognition-related synaptic plasticity. It has been proved by MT2 receptor knockout mice or mice treated with MT2 receptor antagonist. Result showed that MT2 receptor is essential for axonogenesis and axon formation and the downstream of the MT2 receptor consists of Akt/GSK-3β/CRMP-2 cascade (Liu et al. 2015). Apart from that, melatonin was able to reduce electrophysiological degeneration of sciatic nerve in ovariectomized-aged rats. Ovariectomized animal model has been used to mimic women who faced post-menopausal changes, in which the physiological changes have been linked to increased inflammatory markers and oxidative stress in brain (Monteiro et al. 2005; Signorelli et al. 2006; Khodabandehloo et al. 2013). Melatonin-treated rats showed a shorter distal latency and a significantly higher nerve conduction velocity when compared to the untreated rats (Ek et al. 2007). These findings support the potential use of melatonin in clinical application for women with post-menopausal peripheral nerve degeneration (Björkqvist et al. 1977; Pascual et al. 1991; Ek et al. 2007).

The neuroprotective mechanisms of melatonin in PNI are possibly mediated by free radicals scavenging, antioxidative, and analgesic effects. Interestingly, after peripheral axotomy, melatonin reduced dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH)-d/NOS expression on the lesioned hypoglossal neurons (Chang et al. 2000). Moreover, melatonin improved functional recovery of sciatic nerve injury via axonal regeneration, reduced oxidative stress via electron donation to directly detoxify hydroxyl radical, cell-protective effects via mimicking the effects of calcium channel blockers (Atik et al. 2011; Reiter et al. 1997). Melatonin protected neuronal death at low doses (1 to 50 mg/kg), whereas high doses (50 to 100 mg/kg) caused failure to thrive, seizures, or death (Rogério et al. 2002).

Collateral branching and regrowth of axons to incorrect muscles is believed to be the main reasons for poor functional recovery after peripheral nerve lesion. Hence, Guntinas-Lichius et al. suggested that an accurate re-innervation of the neuromuscular junction is required as collateral axonal branching at the lesion site may be the critical limiting factor for the functional recovery after PNI (Guntinas-Lichius et al. 2005). Melatonin has good microsurgical repair during topical application; however, full functional recovery of the injured nerve is never achieved.

Effects of Melatonin on Neurotmesis and Axonotmesis

The effects of melatonin on neurotmesis (cut injury) and axonotmesis (crush injury) were studied several times in the past. Melatonin preserved the structure of myelin sheaths in rats experiencing sciatic nerve cut or sciatic nerve crush. Furthermore, biochemical analyses showed decreased activity of lipid peroxidation and increased activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase in the melatonin-treated rats as compared to vehicle-treated rats (Kaya et al. 2013). Similarly, Atik et al. reported that administration of melatonin after crush and cut injuries could reduce axonal injury, myelin breakdown, and sciatic nerve lipid peroxidation (Atik et al. 2011). It was concluded that supraphysiologic doses of melatonin given over a relatively long period were required to reach such efficacy (Atik et al. 2011).

Stavisky et al. have studied the effects of melatonin on plasmalemmal fusion of sciatic axons in rats severed by crushing injury (Stavisky et al. 2005). Polyethylene glycol (PEG) has been recently used to promote the recovery of crushed sciatic nerve model. The mechanism underlying PEG’s effect has yet to be elaborated, but it is hypothesized that PEG removes water molecules that surrounds the axolemma, leading to aggregation of lipid membranes. This, in turn, allows fusion of lipid bilayer, thus improving recovery of crush-severed injuries (Britt et al. 2010). A significant increase in PEG-induced fusion was shown in sciatic axons of melatonin-treated animals (Stavisky et al. 2005). Hence, this shows the potential use of melatonin in promoting recovery of the sciatic nerve in crush-type injuries (Stavisky et al. 2005). Moreover, Zencirci et al. reported that melatonin increased functional recovery in peripheral nerve crush injury as analyzed using the walking track analysis and sciatic functional index (SFI) (Zencirci et al. 2010). The SFI values were increased in the group treated with 5 mg/kg and 20 mg/kg of melatonin in comparison to the control group.

Effects of Melatonin on Scar Formation

Traditionally, surgeons manage collagen scar formation by close approximation of fascicles of the proximal to distal stumps (Holmes and Young 1942; Fischer et al. 1985). However, complete recovery of nerve function is still not guaranteed in every patient even though great adaptations are made in matching distal and proximal nerve stumps, as well as better guidance are provided in axon regeneration towards the original target tissue (Deumens et al. 2010). Collagen scar formation at the cut end of a peripheral nerve is known to be an important clinical practice for neurosurgeons as it obstructs sprouting of axons into appropriate distal fascicles, and thereby limits the regeneration process. Researchers have attempted to control collagen accumulation and neuroma formation with various physical and chemical methods, but with limited functional success.

A few studies using pinealectomized animals have demonstrated that administration of exogenous melatonin could reduce scar formation in the nerve stump as well as collagen production in granulation tissue at the peripheral nerve (Turgut et al. 2006a). On the other hand, transforming growth factor beta 1 (TGF-β1) and basic fibroblast growth factor (bFGF) are known to stimulate excessive collagen production and scar formation (Heckenkamp et al. 2004; Turgut et al. 2006a). Turgut et al. reported that pinealectomized animals showed an intense immunoreactivity of TGF-β1 and bFGF in the epineurium of animals, whereas negative or weaker positive immunoreactivity could be observed in the melatonin-treated group (Turgut et al. 2006a). Therefore, melatonin might reduce scar formation by reducing the TGF-β1 and bFGF production.

Effects of Melatonin on Cells Degeneration and Apoptosis

MT1 receptor-dependent phosphorylation of extracellular signal-regulated kinases (ERK ½) pathway was found to mediate the effect of melatonin on Schwann cell proliferation (Luchetti et al. 2010;). Activation of ERK pathway was suggested to play a significant role in promoting regeneration of transected nerves at the distal and proximal stumps (Sheu et al. 2000; Agthong et al. 2006). Moreover, Seo et al. suggested that activation of the ERK ½ pathway in Schwann cell proliferation might play a significant role in sciatic nerve regeneration (Seo et al. 2009).

In a recent study using nerve engineering approach, a 3D model of melatonin/polycaprolactone (MLT/PCL) nerve guide conduit showed to promote Schwann cell proliferation and increase expression of peripheral nerve cells markers such as S100, myelin basic protein (MBP), and β-III tubulin (Tuj1) in vitro. Furthermore, it improved in vivo morphological, functional and neural recovery of the peripheral nerves. Other than that, melatonin released from the scaffold enhanced mitochondrial activity, antioxidant property, and anti-inflammatory reactions in PNI. Researchers showed that by using this method, melatonin could be released at a constant rate as melatonin was encapsulated in the PCL scaffold, which slowly degraded in vivo (Qian et al. 2018).

Melatonin is known to inhibit apoptotic cell death in the CNS (Reiter 1998). A study found that melatonin reduced the death of motor neurons caused by sciatic nerve transection in neonatal rats (Rogério et al. 2002). Rogerio et al. found that melatonin administered at doses of 1, 5, 10, and 50 mg/kg before and at seven times interval after sciatic nerve cut injury could significantly decrease the death of motor neurons in the spinal cord of neonatal rats (Rogério et al. 2002). A higher survival rate of neurons was noted in animals treated with a lower dosage of melatonin compared to higher dosage (Rogério et al. 2002). A meta-analysis conducted by Yang et al. showed that melatonin had a therapeutic effect on spinal cord injury where improved neurological recuperation and antioxidant effects could be observed at dose of 12.5 mg/kg on the first day in a rat model (Yang et al. 2016). Apart from that, they reported that melatonin reduced the levels of malondialdehyde, glutathione, and activity of myeloperoxidase.

Conclusion Remarks

Convincing evidence regarding ability of melatonin to cross the blood–brain barrier and its short life with no significant side effects has made melatonin a promising neuroprotective agent. This review highlights the potential neuroprotective effect of melatonin in ischaemia, AD, PD and PNI. Melatonin is clearly an antioxidant, anti-apoptotic, anti-inflammatory, and anti-misfolding molecule. This review has listed out the biological evidence that melatonin increased Schwann cells proliferation, promoted nerve regeneration, decreased cell death, and inhibited scar formation. Melatonin can also inhibit collagen accumulation and neuroma formation as well as enhance healing process and peripheral nerve regeneration. Table 1 and Fig. 2 summarize the possible mechanisms of melatonin in neuroprotection. Nevertheless, further experiments and randomized controlled clinical studies are still required to provide better evidence and insight in terms of the dosage, pharmacokinetics, and pharmacodynamic profiles of melatonin to truly understand its medical potentials in neuroprotection and neuroregeneration.

Table 1.

Summary of representative studies on pharmacological effects of melatonin as neuroprotectant in rodent model

Disease model	Melatonin concentrations	Type of melatonin administration	Duration of treatment	Experimental model	Outcomes	References
Peripheral nerve injury	30 μg/100 g	Melatonin was administered in continuous doses subcutaneously and kept under 12 h light/12 h dark cycle	4 weeks	Male Wistar Rats	Melatonin inhibits neuroma formation and improves axonal regeneration	(Turgut et al. 2005a)
	30 μg/100 g	Melatonin was administered in continuous doses subcutaneously and kept under 12 h light/12 h dark cycle	8 weeks	Male Wistar Rats	Melatonin inhibits neuroma formation and improves axonal regeneration	(Turgut et al. 2005b)
	30 μg/100 g	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	2 months	Female Swiss albino rats	Melatonin reduces the expression of nestin induced by pinealectomy in Cornu ammonis (CA)1 region of the hippocampus	(Turgut et al. 2006b)
	5 mg/kg or 20 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	2 or 6 weeks	Ovariectomized (OVX)-aged Wistar rats	Melatonin treatment increases electrophysiological properties of sciatic nerve	(Ek et al. 2007)
Ischaemia–reperfusion injury	10 mg/kg	Melatonin was administered in a single dose through the tail vein	5 h	Male Wistar rats	Melatonin treatment reduces ischemic degeneration in nerve fibers	(Sayan et al. 2004)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	4 days	Male Mongolian gerbils	Melatonin treatment reduces neuronal loss by inhibiting production of malondialdehyde (MDA) in the brain and myeloperoxidase (MPO) in hippocampus	(Cuzzocrea et al. 2000)
	10 mg/kg, 20 mg/kg, 40 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	24 h, 48 h and 72 h	Male Wistar rats	Melatonin treatment at concentrations of 20 mg/kg and 40 mg/kg reduces the production of malondialdehyde whereas 10 mg/kg shows no effect.	(Sinha et al. 2001)
	Minimum 0.9 pg/ml – 12 pg/mL in daytime	N/A	15 days	Male Sprague–Dawley rats	Endogenous melatonin gives neuroprotective effects; stroke-induced brain injury in pinealectomized rats is more severe than the controls	(Manev et al. 1996)
	5 mg/kg, 15 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	72 h	Male Sprague–Dawley rats	Single injection or multiple injections of melatonin with 5 mg/kg or 15 mg/kg were found to reduce infarct volume	(Pei et al. 2003)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally	15 days	Neonatal Wistar rats	Melatonin treatment decreases level of vascular endothelial growth factor (VEGF), nitric oxide (NO) production and rhodamine isothiocyanate (RhIC) leakage, hence protects the developing hippocampus from hypoxia-associated damage	(Kaur et al. 2008)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	3 weeks	Male Wistar rats	Melatonin treatment reduces peripheral neuropathic pain in streptozotocin-induced diabetic rats	(Babaei-Balderlou et al. 2010)
	10 μg/mL	Melatonin was administered in a single dose via tail vein along with Evans blue injection	2 h	Rat brain microvascular endothelial cells (RBMEC)	Melatonin inhibits matrix metalloproteinase (MMP)-9, attenuates loss of zonula occludens (ZO)-1 junctional integrity and halts F-actin stress fiber formation	(Alluri et al. 2016)
	N/A	N/A	7 weeks	Sprague–Dawley rats	Pinealectomized rats show more working memory errors in the maze compared to control rats	(De Butte et al. 2002)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under a constant temperature of 37 °C ± 0.5 °C	1 week	Male Wistar rats	Preservation of CA1 hippocampal neurons that is treated with melatonin at 0, 2, and 6 h	(Cho et al. 1997)
	5 mg/kg	Melatonin was administered in a single dose intravenously and kept under a constant temperature of 37 °C ± 0.5 °C	72 h	Sprague–Dawley rats	Melatonin treatment improves electrophysiological and neurobehavioral recoveries; melatonin reduces cortical and striatal infarct sizes after cerebral ischemia and reperfusion injury	(Lee et al. 2004)
	2.5 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	15-17 days	Rats	Pinealectomized rats treated with melatonin have reduced injury marker in brain	(Joo et al. 1998a, b)
	4 mg/kg or 8 mg/kg	Melatonin administration method not mentioned	24 h	Wistar rats	Melatonin at 4 mg/kg reduces 40% of infarct volume and improves neurologic deficit scores; increased dose of melatonin to 8 mg/kg shows no significant difference in reducing the infarct volume	(Kilic et al. 1999)
	10 mg/kg	Melatonin was administered in a single dose intraperitoneally and kept under 14 h light/10 h dark cycle	1 h	Mongolian Gerbil	Melatonin inhibits nitric oxide production and scavenges peroxynitrite anion and free radicals	(Guerrero et al. 1997)
	20 μg/mL	Approximately 4 mg/kg of Melatonin was administered orally everyday and kept under 14 h light/10 h dark cycle	20 days	Pregnant Wistar rats	Melatonin prevents ischemia–reperfusion-induced oxidative placental DNA and mitochondrial damage in pregnant rats and preserves fetal growth of rats	(Nagai et al. 2008)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle and maintained a constant temperature of 37 °C	3 h, 6 h, 24 h, 72 h	Sprague–Dawley (SD) rats	Melatonin reduces the expression of ZO-1 and water channels aquaporins-4 (AQP-4); protects neonatal rat model of hypoxic-ischemic brain damage (HIBD) against edema; melatonin treatment reduces the severity of pathology after injury.	(Xu et al. 2017)
	5 mg/kg and 100 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	30 days	Male Wistar rats	Both doses of melatonin suppress NADPH-d/NOS expression, giving antioxidant properties.	(Chang et al. 2000)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	4 weeks and 12 weeks	Female albino rats	Melatonin improved functional recovery of sciatic nerve injury via axonal regeneration, reduced oxidative stress via electron donation.	(Atik et al. 2011)
	1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, and 100 mg/kg	Melatonin was administered in continuous doses subcutaneously	9 days	Wistar rats	Melatonin at doses of 1–50 mg/kg decreased neuronal death whereas doses of 50 to 100 mg/kg caused failure to thrive, seizures, or death.	(Rogério et al. 2002)
	30 μg/100 g	Melatonin was administered in continuous doses subcutaneously and kept under 12 h light/12 h dark cycle	2 months	Wistar rats	Melatonin reduces collagen formation by inhibiting the expression of transforming growth factor beta (TGF β)-1 and/or basic fibroblast growth factor (bFGF)	(Turgut et al. 2006a)
Apoptotic cell death	1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, and 100 mg/kg	Melatonin was administered in continuous doses subcutaneously	4 days	Wistar rats	Melatonin treatment helps decreasing motorneuron death; higher doses of melatonin treatment do not provide better protection than the lower doses; 1–50 mg/kg of melatonin decreases motor neuron death but doses at 50 and 100 mg/kg cause failure to thrive, seizures, or death	(Rogério et al. 2002)
Neurotmesis and axonotmesis	50 mg/kg/day	Melatonin was administrated in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	6 weeks	Female Wistar rats	Melatonin improves sciatic nerve recovery and has demonstrated better structural preservation of myelin sheaths; biochemical analysis also shows a decreased level of lipid peroxidation and higher superoxide dismutase, catalase, and glutathione peroxidase activities	(Kaya et al. 2013)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	4 weeks and 12 weeks	Female albino rats	At 12 weeks, improved functional and electrophysiological of the rats can be observed	(Atik et al. 2011)
	100 μM	Melatonin was applied on lesion site of the sciatic nerve	18 h	Sciatic nerves of Sprague–Dawley rats	Melatonin increases the ability to polyethylene glycol (PEG)-fuse sciatic axons in vivo, compared to control	(Stavisky et al. 2005)
Central nervous system	5 mg/kg and 20 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	21 days	Male Wistar rats	Antioxidant effects of melatonin are found to contribute to nerve recovery where melatonin increases sciatic functional index (SFI) values in injured sciatic nerve, increases conduction velocities and decreases latency values; reduced densities of high frequency components of compound muscle action potential (CMAP) is also reported	(Zencirci et al. 2010)
	0.5 mg/mL	An average of 3 mL/day of melatonin was given orally as drinking water	Initiated 4 months of aged and continued up to 8 months, 9.5 months, 11 months, and 15.5 months	Transgenic mice Tg2576	Mice that received melatonin treatment have enhanced survival rate; melatonin reduces the number of markers of β-amyloid levels and protein nitration.	(Matsubara et al. 2003)
	2.5 mg/kg, 5 mg/kg, and 10 mg/kg	Melatonin was administered in continuous doses subcutaneously and kept under 12 h light/12 h dark cycle	24 h	Sprague–Dawley rats	All doses of melatonin administration increases the expression of neuronal Bcl-2	(Ling et al. 1999)
	20 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	6 days	Male albino BALB/c mice	Melatonin provides neuroprotection of dopaminergic activity in MPTP-treated mice by increasing Tyrosine hydroxylase levels	(Ma et al. 2009)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	5 days	Swiss Webster mice	Melatonin administration provides protection against methamphetamine induced neurotoxicity by increasing the content of striatal dopamine and dopamine transporter binding sites.	(Itzhak et al. 1998)
	2 mg/kg	Melatonin was administered in continuous doses subcutaneously	7 days	Pregnant Wistar rats	Melatonin provides neuroprotection against methamphetamine by increasing the levels of Tyrosine hydroxylase protein, synaptophysin protein and growth-associated protein-43.	(Kaewsuk et al. 2009)
	10 mg/kg	Melatonin was administered in continuous doses subcutaneously and kept under 12 h light/12 h dark cycle	7 days	Wistar rats	Melatonin administration gave neuroprotection against amphetamine by increasing levels of VMAT-2 and phosphorylated tyrosine hydroxylase.	(Mukda et al. 2011)
	10 mg/kg, 20 mg/kg and 30 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	4 days	Male Sprague–Dawley rats	All doses of melatonin administration provide neuroprotection against rotenone by increasing activities of antioxidant enzymes SOD.	(Saravanan et al. 2007)
	10 mg/kg/day	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	14 days	Male Wistar rats	Melatonin administration prevented retinone-induced nigrostriatal neurodegeneration and α-synuclein aggregation.	(Lin et al. 2008)
	1 μmol/L,10 μmol/L, or 100 μmol/L	Melatonin was injected in a single dose into the lateral ventricle of Wistar rats	12 h	Wistar rats	Wortmannin induced in vivo Alzheimer-like hyperphosphorylation of tau was partially inhibited by melatonin with the concentration of 10 μmol/L and 100 μmol/L	(Liu and Wang 2002)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cyle	28 days	Male Wistar rats	Melatonin administration has neuroprotective effects against isoproterenol by inactivating PKA that induces Alzheimer-like abnormal hyperphosphorylation of tau and further promotes oxidative stress.	Wang et al. (2004, 2005a, b, c)
	1 mg/kg or 10 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	10 days	Male Sprague–Dawley rats	Both doses of melatonin administration partially inhibits phosphorylation of Phosphatase-2A, prevent calyculin A-induced synaptophysin loss, memory retention deficits, and hyperphosphorylation of tau and neurofilaments.	(Yang et al. 2011)
	0.1 mg/kg, 1 mg/kg, and 10 mg/kg	Melatonin was administered in continuous doses intraperitoneally with constant light illumination	30 days	Male Wistar rats	Dose of 1 mg and 10 mg of melatonin reverses oxidative stress, tau hyperphosphorylation, spatial memory impairment, and synaptic damages induced by constant light illumination.	(Ling et al. 2009)
	10 mg/kg	Melatonin was administered intraperitoneally to 2 groups of rats which categorized into day group and night group	N/A	Male Sprague–Dawley rats	Administration of melatonin inhibits NFκB, suppressing anti-inflammatory properties.	(Chuang et al. 1996)
	10 mg/kg	Melatonin was administered in continuous doses intraperitoneally under light-phase	4 h	C57/B1 mice	Melatonin reduces lipid peroxidation and increases striatal tyrosine hydroxylase; melatonin provides neuroprotective effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease	(Acuña-Castroviejo et al. 1997)
	500 μg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	35 days	Male C57bl/6 mice	Melatonin prevents cell death and damage induced by chronic administration of MPTP	(Antolín et al. 2002)
	10, 20, and 30 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	48 h	Balb/c mice	All doses of melatonin are found to be able to block MPTP-induced glutathione depletion that leads to oxidative stress in substantia nigra (SN) and nucleus caudatus putamen (NCP)	(Thomas and Mohanakumar 2004)
	5 mg/kg	Melatonin was administered in continuous doses subcutaneously and kept under 12 h light/12 h dark cycle	2 weeks	Sprague–Dawley rats	Prolonged melatonin bioavailability in 6-hydroxydopamine (6-OHDA)-treated rats improves recovery from lesion-induced motor deficits; it also prevents loss in mitochondrial complex I activity and decreases the severity of hemi-Parkinson condition caused by 6-OHDA	(Dabbeni-Sala et al. 2001)
Optic nerve injury	20 mg/kg/day	Melatonin was administered through gavage continuously	4 days, 7 days, 14 days, 21 days, 28 days	Sprague–Dawley rats	Melatonin preserves retinal ganglion cells by reducing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and caspase-3 positive cells	(Wei et al. 2017)
	Pellet of 20 mg with 3% w/v vegetable oil	Melatonin pallet was continuously implanted through subcutaneous route and kept under 12 h light/12 h dark cycle	24 h	Male Wistar rats	Melatonin protects optic nerve by preserving visual evoked potentials (VEPs) and pupil light reflex (PLR) and anterograde transport of cholera toxin β-subunit from the retina to the superior colliculis	(Aranda et al. 2016)
	Initial: 4 mg/kg bw bolus Subsequent 14 days: 8 mg/kg bw/day	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	14 days	Male C57BL/6	Exogenous melatonin administration increases the density of retinal ganglion cells after optic nerve transection in pinealectomized mice	(Kilic et al. 2002)
	5 mg/kg/day	Melatonin was administered in continuous doses intraperitoneally	7 days, 14 days, 30 days	Sprague–Dawley rats	Melatonin inhibits apoptosis of retinal ganglion cells after ischemia reperfusion injury; melatonin improves the survival rate of retinal ganglion cells after ischemic-reperfusion injury	(Tang et al. 2006)
	5 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	24 h and 96 h	Female Wistar rats	Reduced retinal swelling is observed in melatonin-treated rats, however the hyperchromatic nuclei and apoptotic ganglion cells are still greater than normal; melatonin also protects retinal from nitric oxide-induced cellular changes within the first day but the effect does not last over 4 days	(Siu et al. 2004)
	40 mg/kg	Melatonin was administered in continuous doses intraperitoneally and kept under 12 h light/12 h dark cycle	2 weeks	C57BL/6 mice	Melatonin inhibits expression of hypoxia-inducible factor -alpha; melatonin increases the survival rate of retinal ganglion cells	(Park et al. 2012)

Fig. 2.

Summary of possible mechanisms of melatonin in neuroprotection

Author Contributions

HYT wrote the manuscript, and KYN and RYK critically reviewed the manuscript. SMC formulated the entire concept and reviewed the manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Research Involving Human Participants or Animals

This article does not contain any studies with human participants or animals performed by any of the authors.