Int. J. Mol. Sci. 2013, 14, 9379-9395; doi:10.

3390/ijms14059379
OPEN ACCESS
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review

Neuroprotective Effect of Melatonin: A Novel Therapy against

Perinatal Hypoxia-Ischemia
Daniel Alonso-Alconada *, Antonia Álvarez, Olatz Arteaga, Agustín Martínez-Ibargüen and
Enrique Hilario

Department of Cell Biology and Histology, School of Medicine and Dentistry,

University of the Basque Country, Barrio Sarriena s/n, Leioa 48940, Bizkaia, Spain;
E-Mails: antoniaangeles.alvarez@ehu.es (A.A.); olatz_b@hotmail.com (O.A.);
agustin.martinez@ehu.es (A.M.-I.); enrique.hilario@ehu.es (E.H.)

* Author to whom correspondence should be addressed; E-Mail: daniel.alonsoa@ehu.es;

Tel.: +34-946-013-294 (ext. 123); Fax: +34-946-013-266.

Received: 28 February 2013; in revised form: 15 April 2013 / Accepted: 16 April 2013 /
Published: 29 April 2013

Abstract: One of the most common causes of mortality and morbidity in children is
perinatal hypoxia-ischemia (HI). In spite of the advances in neonatology, its incidence is
not diminishing, generating a pediatric population that will require an extended amount of
chronic care throughout their lifetime. For this reason, new and more effective
neuroprotective strategies are urgently required, in order to minimize as much as possible
the neurological consequences of this encephalopathy. In this sense, interest has grown in
the neuroprotective possibilities of melatonin, as this hormone may help to maintain cell
survival through the modulation of a wide range of physiological functions. Although some
of the mechanisms by which melatonin is neuroprotective after neonatal asphyxia remain a
subject of investigation, this review tries to summarize some of the most recent advances
related with its use as a therapeutic drug against perinatal hypoxic-ischemic brain injury,
supporting the high interest in this indoleamine as a future feasible strategy for cerebral
asphyctic events.

Keywords: melatonin; neuroprotection; hypoxia-ischemia; newborn

Int. J. Mol. Sci. 2013, 14 9380

1. Introduction

The main function of the pineal gland in all species is to transduce information concerning
light-dark cycles to body physiology, particularly for organization of body rhythms via its main
hormone melatonin [1]. Based on the ability of melatonin (N-acetyl-5-methoxytryptamine) and its
metabolites to scavenge a wide variety of free radicals (FR), it is not surprising to consider it as one of
its most important functions in living organisms leading to protect them from oxidative stress [2,3].
Acting as a direct scavenger, this neurohormone is able to remove FR, such as singlet oxygen,
superoxide anion radical, hydroperoxide, hydroxyl radical and the lipid peroxide radical [2,4].
Moreover, a single melatonin molecule may generate products in a scavenger cascade, which may
collectively eliminate up to ten FR [4]. Melatonin can develop indirect antioxidant actions through the
improvement of the mitochondrial efficiency [5], the stimulation of the gene expression and the
activation of some of the most important antioxidant enzymes, including superoxide dismutase (SOD),
catalase, glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione peroxidase [6] and
also with the strengthening of the antioxidant effect of substances, like glutathione, vitamin E and
vitamin C [7].
The brain is particularly sensitive to FR damage due to its high utilization of oxygen, its relatively
poorly developed antioxidant defense and its high amount of easily oxidizable fatty acids. Thus, the
use of melatonin as pharmacological agent against neurodegenerative disorders, such as Huntington's
disease, Alzheimer’s disease and Parkinsonism and also against ischemic brain injury/stroke, has been
extensively evaluated. With an incidence of 2–6/1,000 term births [8], perinatal hypoxia-ischemia (HI)
remains the single most important cause of brain injury in the newborn, leading to death or lifelong
disability [9,10]. Despite the improvements in perinatal care, significant neurological sequelae can
occur in as many as 50%–75% of these asphyctic children, which may suffer from long-term
neurological consequences, such as cerebral palsy, mental retardation and epilepsy [11–13]. Asphyxia
is also associated with attention deficits and hyperactivity in children and adolescents [14,15]. During
the last decade, melatonin has started to be considered as an attractive option in order to minimize as
much as possible the neurological sequelae from hypoxic-ischemic brain injury. It easily crosses both
the placental and the blood-brain barrier, reaching subcellular compartments with a low toxicity and
high efficacy [16–18], and even at high supra-physiological concentrations, there appear to be no
adverse side-effects [19], making it a relatively-safe therapy that could be administered to babies.

2. Brain Protection

Following cerebral asphyxia, HI starts out a multi-faceted cascade of events that ultimately causes
cell death and often damages the whole brain [20]. Everything begins when the reduction in oxygen
and blood supply induces a decrease in oxidative phosphorylation and the neonate’s brain converts to
anaerobic metabolism in an effort to sustain functional ability. Anaerobic metabolism leads to a 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. Afterwards, multiple and diverse downstream biochemical
reactions aggravate the pathogenesis of hypoxic-ischemic brain damage, being the most important,
among others, the production of reactive oxygen species leading to oxidative stress, the massive increase
Int. J. Mol. Sci. 2013, 14 9381

in free cytosolic calcium concentrations and the drop in mitochondrial function triggering the
activation of apoptotic pathways, DNA fragmentation and cell death. After ischemic brain injury/stroke,
melatonin has showed a remarkable capacity to reduce infarct volume and/or inhibit neuronal cell
death after in different mammalian species and using different experimental models [21–33].

Figure 1. Nissl-stained (A–C), myelin basic protein (D–F) and glial fibrillary acidic
protein (G–I) immunolabeled brain sections corresponding to the surrounding areas of the
CA1 region of the hippocampus and the external capsule showing cell loss (B),
myelination deficit (E) and reactive gliosis (H) after hypoxia-ischemia and recovery after
melatonin administration. Seven-day old rats were subjected to hypoxia-ischemia
(left common carotid artery ligated and then 8% oxygen for 2 h) and sacrificed seven days
after the injury. Pups without ischemia or hypoxia served as controls (Sham group).
Bar: 100 m.
Int. J. Mol. Sci. 2013, 14 9382

Melatonin administration after neonatal HI has been shown to reduce infarct volume both
administered before or after the injury [34–36]. Indeed, melatonin was able to decrease sensorimotor
asymmetry and learning deficits, thus protecting the pups from the long-term consequences of neonatal
asphyxia [34]. While virtually every cell is affected by asphyxia, they do not respond in the same way
during HI, being neurons the most sensitive cells to the lack of oxygen and showing a selective
vulnerability [37–39]. Histological analysis demonstrated an increase in the number of
morphologically well preserved neurons in melatonin-treated animals in the CA1, CA2–CA3 areas and
dentate gyrus of the hippocampus and parietal cortex when compared with the hypoxic-ischemic
group [33,40,41] (Figure 1). Even though astrocytes are more resistant than neurons to oxygen
deprivation, their death may give rise to a new wage of neuronal death due to their role in the
maintenance of the homeostatic state of neurons. Therefore, astrocytes can modulate in a significant
manner the extension and degree of severity of the damage [42,43], either conferring neuroprotection
by scavenging reactive oxygen species and also assisting with reconstruction from brain injury [44] or
leading to deficiencies in the myelination processes, neuronal signaling impairment and an increase the
inflammatory response [45,46]. Melatonin has demonstrated to reduce the expression of the glial
fibrillary acidic protein [33] (Figure 1), whose accumulation is related with the creation of new
astrocytic processes and reactive gliosis [43]. In addition to neurons, oligodendrocytes are particularly
vulnerable to asphyxia, affecting myelination that gives rise to white matter lesions and damaging gray
matter oligodendrocyte progenitors [47]. An abnormal decrease in the expression of myelin basic
protein leading to myelination deficit is considered hallmark of inflammation-associated diffuse white
matter damage [48,49]. In this sense, several groups have suggested that melatonin may be of
therapeutic value in ameliorating hypoxic-ischemic damage to the developing white matter through
normalization of the myelination process [33,50–52] (Figure 1).

3. Antioxidant

The high incidence of hypoxic-ischemic brain lesions in newborns can be partly attributed to the
fact that the developing brain is especially vulnerable to oxidative stress. After neonatal asphyxia,
reperfusion brings about the overproduction of FR leading to oxidative stress, as the antioxidant
capacity of immature neurons is easily overwhelmed by hypoxia-induced reactive oxygen species. The
newborn brain is especially vulnerable to oxidative imbalance, due to its increased fatty acid content,
higher concentrations of free iron, high rates of oxygen consumption, low concentrations of antioxidant,
an imbalance of antioxidant enzymes, as for example catalase CuZn-SOD-1, mitochondrial SOD-2 and
glutathione peroxidase and oxygen-induced vasoconstriction, leading to reduced brain perfusion,
among others [53–55].
When membrane lipoproteins and polyunsaturated fatty acids suffer attacks from FR, many
oxygenated compounds, particularly aldehydes, such as malondialdehyde (MDA), are produced. Thus,
the evaluation of lipid peroxidation is a useful tool to evaluate oxidative stress leading to brain
damage. Melatonin has been able to abolish lipid peroxidation in late-gestation fetal sheep in respond
to umbilical cord occlusion [56] and to avoid the rise in MDA induced by hypoxia in rat pups [57] and
asphyxiated human newborns [58]. Deferoxamine-chelatable free iron, isoprostanes, neuroprostanes
and neurofurans are also quantitative biomarkers of oxidative damage [59–61], and after melatonin
Int. J. Mol. Sci. 2013, 14 9383

administration, their levels were significantly lower than those in hypoxic-ischemic rats [62,63]. These
results were similar to those observed in fetal sheep after umbilical cord occlusion, where the
production of 8-isoprostanes was attenuated [64]. On the other hand, melatonin may prevent protein
oxidation in the brain tissue of hypoxic neonatal rats [65], as terminal products of protein exposure to
FR are considered reliable markers of the degree of protein damage in oxidative stress [66].
Additionally, the activity of the antioxidative enzyme catalase is maintained [57], hydroxyl formation
reduced [56] and nitrite/nitrate levels reduced [58] in different animals models subjected to hypoxia.

4. Anti-Apoptotic

Under pathophysiological conditions, one of the most important key regulators of apoptotic cell
death is mitochondrial impairment, as the disruption of its membrane integrity and loss of membrane
potential can determinate cell survival by overproduction of reactive oxygen species, abnormal
calcium homeostasis and release of apoptotic proteins. In several studies demonstrating anti-apoptotic
actions, melatonin prevented cytochrome c release [32,67,68], reduced or blocked caspase-1
and caspase-3 activation [32,67,69–73], increased the expression of anti-apoptotic proteins
Bcl-2 [71,74,75] and Bcl-xL[70], diminished Bad [31,72] and Bax [71] pro-apoptotic proteins,
inhibited poly-ADP-ribose-polymerase cleavage [72], avoided mitochondrial permeability transition
pore opening, thus counteracting the collapse of the mitochondrial membrane potential [67,69], and
decreased the number of TUNEL-positive cells/DNA breaks [31,32,72,75–78]. In the central nervous
system, melatonin can also generate anti-excitatory effects on neurons through the modulation of
gamma-aminobutyric acid and glutamate receptors [79,80], inducing a decrease in cytosolic calcium
concentrations [81,82].
Shortly after a hypoxic-ischemic event, reactive oxygen species overproduction can start out a
harmful multi-faceted cascade that includes lipid peroxidation, protein oxidation and DNA
fragmentation, ultimately damaging vital cellular components as nucleic acids, cell membranes and
mitochondria, resulting in subsequent cell death in the immature brain [83–85]. In this regard, we have
recently shown that HI can develop a widespread increase in reactive oxygen species after perinatal
asphyxia, an overproduction correlated with the number of, as well as with the distribution of apoptotic
cells [86].
Melatonin may be an effective prophylactic agent for use in late pregnancy to protect against
mitochondrial-induced cell death after a hypoxic-ischemic event at birth. Given to pregnant rats, it
prevented oxidative mitochondria damage after ischemia-reperfusion in premature fetal rat brain [87]
by means of the maintenance of the number of intact mitochondria and the respiratory control index
(an indicator of mitochondrial respiratory activity), as well as the reduction in thiobarbituric
acid-reactive substances concentration (a marker of oxidative stress) [40,41]. Hutton et al. studied
caspase-3 activation and fractin in order to evaluate the anti-apoptotic effect of melatonin in a model of
birth asphyxia in the spiny mouse, showing lower levels after its administration [88]. Accordingly,
Fu et al. demonstrated the inhibition of caspase-3 activation, the induction of Bcl-2 expression and the
increase Bcl-2/Bax ratio in a model of hypoxia in vitro [89]. Melatonin administration not only
generates a neuroprotective effect when administered before the hypoxic-ischemic event, but also
when given after the onset of the injury. Using the terminal deoxynucleotidyl transferase dUTP nick
Int. J. Mol. Sci. 2013, 14 9384

end labeling method (TUNEL) to detect DNA fragmentation and apoptotic figures, melatonin-treated
neonatal animals have shown a reduction in TUNEL-positive cells per unit area in neonatal sheep [64]
and in rats [33,35,36].
As shown above, melatonin can exert a wide range of antiapoptotic effects, mainly targeting
mitochondria, but it can also enhance cell survival pathways leading to cell rescue. For instance,
protection from cerebral ischemic injury was attributed to the maintenance of signaling via the MAP
kinase pathway, leading to the prevention Bad dephosphorylation [31]. Furthermore, melatonin can
target PI3K/Akt pathway [70,72,90–92], mTOR [93] or the forkhead transcription factor, pAFX [91],
and also restore JNK1/2 and ERK 1/2 phosphorylated levels [72,90], thereby preventing the
proapoptotic actions of the dephosphorylated proteins.

5. Anti-Inflammatory

In recent years, the use of melatonin has started to be considered as another meaningful tool against
inflammatory response in an effort to improve the clinical course of illnesses, which have an
inflammatory etiology. The strategy mediated by melatonin and its main metabolites 6-hydroxy,
N1-acetyl-N2-formyl-5-metho, N1-acetyl-5-methoxykynuramine and cyclic 3-hydroxy melatonin,
encompasses the downregulation of some inflammation-related molecules, such as cytokines
interleukin-6, interleukin-8 and tumor necrosis factor- [94–100], 5-lipoxygenase [101],
cyclooxygenase [102,103] and prostaglandin [104–106], an important reduction of nitric oxide (NO)
and MDA levels [107] and also the inhibition of neuronal (nNOS) [108–111] and inducible (iNOS)
nitric oxide synthases [111,112]. Even though NO participates in diverse processes acting as a
physiological messenger, an excess in its concentrations can induce energy depletion, liberation of
excitotoxic amino acids and a high ability to react with other FR. Downregulation of nNOS may
contribute to the maintenance of electron transport chain function [109,110,113–115], thus protecting
from NO-mediated mitochondrial impairment and cell damage [116–119]. From its part, counteraction
to iNOS can avoid lipid peroxidation, shifts the glutathione redox state and boosts energy efficiency
and ATP production in mitochondria [118,120,121]. Regarding the field of ischemic brain injury,
melatonin and its metabolites have been able to reverse the inflammatory response and edema after
stroke suppressing the production of inflammatory cytokines [102,122,123], reducing NOS [124],
preventing the translocation of NF- B to the nucleus [125,126] and decreasing cyclooxygenase-2 gene
expression [127], molecular changes correlated with a reduction in the size of brain infarcts.
After perinatal asphyxia, FR also stimulate ischemic cells to secrete inflammatory cytokines and
chemokines, which in turn can generate a wide variety of cytotoxic agents, including more cytokines,
matrix metalloproteases, NO and more reactive oxygen species. These molecules can dismantle both
blood brain barrier and extracellular matrix, allowing the blood, soluble elements and peripheral
inflammatory cells to penetrate the brain, resulting in the exacerbation of the damage. Melatonin may
be beneficial, as it reduces NO production, vascular endothelial growth factor concentration and,
hence, vascular permeability that results increased after hypoxic exposure [128]. Prophylactic maternal
treatment with melatonin has also demonstrated a reduction in central nervous system
inflammation, by limiting macrophage infiltration and glial cell activation in a model of birth asphyxia
in the spiny mouse [88]. Indeed, a reduced number of ED1 positive cells, a marker of activated
Int. J. Mol. Sci. 2013, 14 9385

microglia-macrophages, was found in neonatal rats treated with melatonin when comparing with pups
without treatment [63].

Table. Summary of the experimental evidence regarding the beneficial effects of melatonin.
Target Effect References
Brain Protection
Infarct volume [34–36]
Sensorimotor asymmetry [34]
Learning deficits [34]
Morphologically well preserved neurons [33,40,41]
GFAP expression [33]
MBP expression [33,50–52]
Antioxidant
Lipid peroxidation and MDA production [56–58]
Iso- and neuroprostanes and neurofurans [62–64]
Protein oxidation [65]
Catalase’s activity [57]
Hydroxyl formation [56]
Nitrite/nitrate levels [58]
Anti-apoptotic
Cytochrome c release [32,67,68]
Caspase-1 and Caspase-3 activation [32,67,69–73,88,89]
Bcl-xL and Bcl-2 expression [70,71,74,75,89]
Bax expression [71]
Poly-ADP-ribose-polymerase cleavage [72]
Mitochondrial transition pore opening [67,69]
TUNEL-positive cells/DNA breaks [31–33,35,36,64,72,75–78]
Cytosolic calcium concentrations [81,82]
Oxidative mitochondria damage [87]
Mitochondrial respiratory activity [40,41]
Oxidative stress [40,41]
Fractin levels [88]
Bcl-2/Bax ratio [89]
MAP kinase, JNK1/2 and ERK 1/2 [31,72,90]
Bad dephosphorylation [31]
Anti-inflammatory
Interleukin-6, Interleukin -8 and Tumor Necrosis Factor- [94–100,102,122,123,125,126]
5-lipoxygenase and Cyclooxyenase-2 [101–103,127]
Prostaglandin [104,106]
NO, nNOS, iNOS and VEGF [107–112,124,128]
Macrophage infiltration [88]
ED1 positive cells [63]
Int. J. Mol. Sci. 2013, 14 9386

6. Conclusions

Nowadays, there are convincing evidences demonstrating that melatonin treatment is highly
effective against hypoxic-ischemic brain injury in different animal models by reducing infarct volume
and neuronal loss, minimizing lipid and protein peroxidation, blocking some apoptotic pathways,
inhibiting FR production and decreasing inflammation. Melatonin supplementation, which has a
benign safety profile, may help to reduce complications in the neonatal period that are associated with
short gestation [129] and has demonstrated not only neuroprotective actions against HI in animal
models, but also in preliminary clinical trials [57,130–132].
Nevertheless, the complexity of neonatal hypoxic-ischemic pathophysiology determines that
successful neuroprotection could be achieved only by multi-therapeutic approaches and optimizing
therapy for neonatal brain injury will require capitalizing on multiple pathways, which prevent cell
death. The use of synergic strategies, such as the association between hypothermia and other
therapeutic drugs, may lead to a larger neuroprotective effect on the brain thus improving the neonatal
outcome. In this regard, Robertson et al. have recently shown that melatonin administration to
newborn piglets augments hypothermic neuroprotection by improving cerebral energy metabolism and
by reducing brain damage [133].
Melatonin’s protective actions include not only its direct free radical scavenging, but also the
interaction of its receptors and several yet-undefined functions, so the mechanisms underlying its
neuroprotective benefits are not yet fully elucidated. Moreover, its variable oral absorption and rapid
metabolization [134–136], the search for an appropriate dosage to obtain an antioxidant effect without
desensitize melatonin receptors and its different pharmacokinetic profile when comparing preterm
infants with adults (the half-life of melatonin in neonates is approximately 15 h, while in adults, it is
around 45–60 min) [137], highlight the work that needs to be done before melatonin comes into
clinical practice in a neonatal or pediatric critical care unit.

Conflict of Interest

The authors declare no conflict of interest.

References

1. Arendt, J. Melatonin and the Mammalian Pineal Gland; Chapman & Hall: London, UK, 1995.
2. Tan, D.X.; Manchester, L.C.; Terron, M.P.; Flores, L.J.; Reiter, R.J. One molecule, many
derivatives: A never-ending interaction of melatonin with reactive oxygen and nitrogen species?
J. Pineal Res. 2007, 42, 28–42.
3. Tan, D.X.; Hardeland, R.; Manchester, L.C.; Paredes, S.D.; Korkmaz, A.; Sainz, R.M.;
Mayo, J.C.; Fuentes-Broto, L.; Reiter, R.J. The changing biological roles of melatonin during
evolution: From an antioxidant to signals of darkness, sexual selection and fitness. Biol. Rev.
Camb. Philos. Soc. 2010, 85, 607–623.
Int. J. Mol. Sci. 2013, 14 9387

4. Rosen, J.; Than, N.N.; Koch, D.; Poeggeler, B.; Laatsch, H.; Hardeland, R. Interactions of
melatonin and its metabolites with the ABTS cation radical: Extension of the radical scavenger
cascade and formation of a novel class of oxidation products, C2-substituted 3-indolinones.
J. Pineal Res. 2006, 41, 374–381.
5. Acuna-Castroviejo, D.; Martin, M.; Macias, M.; Escames, G.; Leon, J.; Khaldy, H.; Reiter, R.J.
Melatonin, mitochondria, and cellular bioenergetics. J. Pineal Res. 2001, 30, 65–74.
6. Tomas-Zapico, C.; Coto-Montes, A. A proposed mechanism to explain the stimulatory effect of
melatonin on antioxidative enzymes. J. Pineal Res. 2005, 39, 99–104.
7. Reiter, R.J.; Tan, D.X.; Osuna, C.; Gitto, E. Actions of melatonin in the reduction of oxidative
stress. A Review. J. Biomed. Sci. 2000, 7, 444–458.
8. De Haan, M.; Wyatt, J.S.; Roth, S.; Vargha-Khadem, F.; Gadian, D.; Mishkin, M. Brain and
cognitive-behavioural development after asphyxia at term birth. Dev. Sci. 2006, 9, 350–358.
9. Du Plessis, A.J.; Volpe, J.J. Perinatal brain injury in the preterm and term newborn. Curr. Opin.
Neurol. 2002, 15, 151–157.
10. Hamrick, S.E.; Ferriero, D.M. The injury response in the term newborn brain: Can we
neuroprotect? Curr. Opin. Neurol. 2003, 16, 147–154.
11. Volpe, J.J. Perinatal brain injury: From pathogenesis to neuroprotection. Ment. Retard. Dev.
Disabil. Res. Rev. 2001, 7, 56–64.
12. Low, J.A. Determining the contribution of asphyxia to brain damage in the neonate. J. Obstet.
Gynaecol. Res. 2004, 30, 276–286.
13. Vannucci, S.J.; Hagberg, H. Hypoxia-ischemia in the immature brain. J. Exp. Biol. 2004, 207,
3149–3154.
14. Maneru, C.; Junque, C.; Botet, F.; Tallada, M.; Guardia, J. Neuropsychological long-term
sequelae of perinatal asphyxia. Brain Inj. 2001, 15, 1029–1039.
15. Yager, J.Y.; Armstrong, E.A.; Black, A.M. Treatment of the term newborn with brain injury:
Simplicity as the mother of invention. Pediatr. Neurol. 2009, 40, 237–243.
16. Vitte, P.A.; Harthe, C.; Lestage, P.; Claustrat, B.; Bobillier, P. Plasma, cerebrospinal fluid, and
brain distribution of 14C-melatonin in Rat: A biochemical and autoradiographic study. J. Pineal
Res. 1988, 5, 437–453.
17. Menendez-Pelaez, A.; Reiter, R.J. Distribution of melatonin in mammalian tissues: The relative
importance of nuclear versus cytosolic localization. J. Pineal Res. 1993, 15, 59–69.
18. Gupta, Y.K.; Gupta, M.; Kohli, K. Neuroprotective role of melatonin in oxidative stress
vulnerable brain. Indian J. Physiol. Pharmacol. 2003, 47, 373–386.
19. Rees, S.; Harding, R.; Walker, D. The biological basis of injury and neuroprotection in the fetal
and neonatal brain. Int. J. Dev. Neurosci. 2011, 29, 551–563.
20. Hilario, E.; Alvarez, A.; Alvarez, F.J.; Gastiasoro, E.; Valls-i-Soler, A.; Cellular mechanisms in
perinatal hypoxic-ischemic brain injury. Curr. Pediatr. Rev. 2006, 2, 131–141.
21. Manev, H.; Uz, T.; Kharlamov, A.; Joo, J.Y. Increased brain damage after stroke or excitotoxic
seizures in melatonin-deficient rats. FASEB J. 1996, 10, 1546–1551.
22. Li, X.J.; Zhang, L.M.; Gu, J.; Zhang, A.Z.; Sun, F.Y. Melatonin decreases production of
hydroxyl radical during cerebral ischemia-reperfusion. Zhongguo Yao Li Xue Bao 1997, 18,
394–396.
Int. J. Mol. Sci. 2013, 14 9388

23. Cho, S.; Joh, T.H.; Baik, H.H.; Dibinis, C.; Volpe, B.T. Melatonin administration protects CA1
hippocampal neurons after transient forebrain ischemia in rats. Brain Res. 1997, 755, 335–338.
24. Kilic, E.; Ozdemir, Y.G.; Bolay, H.; Kelestimur, H.; Dalkara, T. Pinealectomy aggravates and
melatonin administration attenuates brain damage in focal ischemia. J. Cereb. Blood Flow
MeTable 1999, 19, 511–516.
25. Wakatsuki, A.; Okatani, Y.; Izumiya, C.; Ikenoue, N. Melatonin protects against ischemia and
reperfusion-induced oxidative lipid and DNA damage in fetal rat brain. J. Pineal Res. 1999, 26,
147–152.
26. Joo, J.Y.; Uz, T.; Manev, H. Opposite effects of pinealectomy and melatonin administration on
brain damage following cerebral focal ischemia in rat. Restor. Neurol. Neurosci. 1998, 13,
185–191.
27. Cuzzocrea, S.; Costantino, G.; Gitto, E.; Mazzon, E.; Fulia, F.; Serraino, I.; Cordaro, S.;
Barberi, I.; De Sarro, A.; Caputi, A.P. Protective effects of melatonin in ischemic brain injury.
J. Pineal Res. 2000, 29, 217–227.
28. Letechipia-Vallejo, G.; Gonzalez-Burgos, I.; Cervantes, M. Neuroprotective effect of melatonin
on brain damage induced by acute global cerebral ischemia in cats. Arch. Med. Res. 2001, 32,
186–192.
29. Zhang, J.; Guo, J.D.; Xing, S.H.; Gu, S.L.; Dai, T.J. The protective effects of melatonin on global
cerebral ischemia-reperfusion injury in gerbils. Yao Xue Xue Bao 2002, 37, 329–333.
30. Pei, Z.; Pang, S.F.; Cheung, R.T. Administration of melatonin after onset of ischemia reduces the
volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model. Stroke 2003,
34, 770–775.
31. Koh, P.O. Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation
of pBad from 14-3-3. J. Pineal Res. 2008, 44, 101–106.
32. Wang, X.; Figueroa, B.E.; Stavrovskaya, I.G.; Zhang, Y.; Sirianni, A.C.; Zhu, S.; Day, A.L.;
Kristal, B.S.; Friedlander, R.M. Methazolamide and melatonin inhibit mitochondrial cytochrome
C release and are neuroprotective in experimental models of ischemic injury. Stroke 2009, 40,
1877–1885.
33. Alonso-Alconada, D.; Alvarez, A.; Lacalle, J.; Hilario, E. Histological study of the protective
effect of melatonin on neural cells after neonatal hypoxia-ischemia. Histol. Histopathol. 2012,
27, 771–783.
34. Carloni, S.; Perrone, S.; Buonocore, G.; Longini, M.; Proietti, F.; Balduini, W. Melatonin
protects from the long-term consequences of a neonatal hypoxic-ischemic brain injury in rats.
J. Pineal Res. 2008, 44, 157–164.
35. Cetinkaya, M.; Alkan, T.; Ozyener, F.; Kafa, I.M.; Kurt, M.A.; Koksal, N. Possible
neuroprotective effects of magnesium sulfate and melatonin as both pre- and post-treatment in a
neonatal hypoxic-ischemic rat model. Neonatology 2011, 99, 302–310.
36. Ozyener, F.; Cetinkaya, M.; Alkan, T.; Goren, B.; Kafa, I.M.; Kurt, M.A.; Koksal, N.
Neuroprotective effects of melatonin administered alone or in combination with topiramate in
neonatal hypoxic-ischemic rat model. Restor. Neurol. Neurosci. 2012, 30, 435–444.
37. Mattson, M.P.; Guthrie, P.B.; Kater, S.B. Intrinsic factors in the selective vulnerability of
hippocampal pyramidal neurons. Prog. Clin. Biol. Res. 1989, 317, 333–351.
Int. J. Mol. Sci. 2013, 14 9389

38. Johnston, M.V. Selective vulnerability in the neonatal brain. Ann. Neurol. 1998, 44, 155–156.
39. Northington, F.J.; Ferriero, D.M.; Graham, E.M.; Traystman, R.J.; Martin, L.J. Early
neurodegeneration after hypoxia-ischemia in neonatal rat is necrosis while delayed neuronal
death is apoptosis. Neurobiol. Dis. 2001, 8, 207–219.
40. Hamada, F.; Watanabe, K.; Wakatsuki, A.; Nagai, R.; Shinohara, K.; Hayashi, Y.; Imamura, R.;
Fukaya, T. Therapeutic effects of maternal melatonin administration on ischemia/reperfusion-induced
oxidative cerebral damage in neonatal rats. Neonatology 2010, 98, 33–40.
41. Watanabe, K.; Hamada, F.; Wakatsuki, A.; Nagai, R.; Shinohara, K.; Hayashi, Y.; Imamura, R.;
Fukaya, T. Prophylactic administration of melatonin to the mother throughout pregnancy can
protect against oxidative cerebral damage in neonatal rats. J. Matern. Fetal. Neonatal Med. 2012,
25, 1254–1259.
42. Takuma, K.; Baba, A.; Matsuda, T. Astrocyte apoptosis: Implications for neuroprotection.
Prog. Neurobiol. 2004, 72, 111–127.
43. Panickar, K.S.; Norenberg, M.D. Astrocytes in cerebral ischemic injury: Morphological and
general considerations. Glia 2005, 50, 287–298.
44. Sizonenko, S.V.; Camm, E.J.; Dayer, A.; Kiss, J.Z. Glial responses to neonatal hypoxic-ischemic
injury in the rat cerebral cortex. Int. J. Dev. Neurosci. 2008, 26, 37–45.
45. Huang, Z.; Liu, J.; Cheung, P.Y.; Chen, C. Long-term cognitive impairment and myelination
deficiency in a rat model of perinatal hypoxic-ischemic brain injury. Brain Res. 2009, 1301,
100–109.
46. Xiong, M.; Yang, Y.; Chen, G.Q.; Zhou, W.H. Post-ischemic hypothermia for 24h in P7 rats
rescues hippocampal neuron: Association with decreased astrocyte activation and inflammatory
cytokine expression. Brain Res. Bull. 2009, 79, 351–357.
47. Rothstein, R.P.; Levison, S.W. Gray matter oligodendrocyte progenitors and neurons die
caspase-3 mediated deaths subsequent to mild perinatal hypoxic/ischemic insults. Dev. Neurosci.
2005, 27, 149–159.
48. Inder, T.E.; Wells, S.J.; Mogridge, N.B.; Spencer, C.; Volpe, J.J. Defining the nature of the
cerebral abnormalities in the premature infant: A qualitative magnetic resonance imaging study.
J. Pediatr. 2003, 143, 171–179.
49. Wang, X.; Hagberg, H.; Zhu, C.; Jacobsson, B.; Mallard, C. Effects of intrauterine inflammation
on the developing mouse brain. Brain Res. 2007, 1144, 180–185.
50. Olivier, P.; Fontaine, R.H.; Loron, G.; van Steenwinckel, J.; Biran, V.; Massonneau, V.;
Kaindl, A.; Dalous, J.; Charriaut-Marlangue, C.; Aigrot, M.S. et al. Melatonin promotes
oligodendroglial maturation of injured white matter in neonatal rats. PLoS One 2009, 4, e7128.
51. Kaur, C.; Sivakumar, V.; Ling, E.A. Melatonin protects periventricular white matter from
damage due to hypoxia. J. Pineal Res. 2010, 48, 185–193.
52. Villapol, S.; Fau, S.; Renolleau, S.; Biran, V.; Charriaut-Marlangue, C.; Baud, O. Melatonin
promotes myelination by decreasing white matter inflammation after neonatal stroke. Pediatr. Res.
2011, 69, 51–55.
53. McLean, C.; Ferriero, D. Mechanisms of hypoxic-ischemic injury in the term infant.
Semin. Perinatol. 2004, 28, 425–432.
Int. J. Mol. Sci. 2013, 14 9390

54. Sheldon, R.A.; Jiang, X.; Francisco, C.; Christen, S.; Vexler, Z.S.; Tauber, M.G.; Ferriero, D.M.
Manipulation of antioxidant pathways in neonatal murine brain. Pediatr. Res. 2004, 56, 656–662.
55. McQuillen, P.S.; Ferriero, D.M. Selective vulnerability in the developing central nervous system.
Pediatr. Neurol. 2004, 30, 227–235.
56. Miller, S.L.; Yan, E.B.; Castillo-Melendez, M.; Jenkin, G.; Walker, D.W. Melatonin provides
neuroprotection in the late-gestation fetal sheep brain in response to umbilical cord occlusion.
Dev. Neurosci. 2005, 27, 200–210.
57. Tutunculer, F.; Eskiocak, S.; Basaran, U.N.; Ekuklu, G.; Ayvaz, S.; Vatansever, U. The
protective role of melatonin in experimental hypoxic brain damage. Pediatr. Int. 2005, 47,
434–439.
58. Fulia, F.; Gitto, E.; Cuzzocrea, S.; Reiter, R.J.; Dugo, L.; Gitto, P.; Barberi, S.; Cordaro, S.;
Barberi, I. Increased levels of malondialdehyde and nitrite/nitrate in the blood of asphyxiated
newborns: Reduction by melatonin. J. Pineal Res. 2001, 31, 343–349.
59. Kohen, R.; Nyska, A. Oxidation of biological systems: Oxidative stress phenomena, antioxidants,
redox reactions, and methods for their quantification. Toxicol. Pathol. 2002, 30, 620–650.
60. Arneson, K.O.; Roberts, L.J., 2nd. Measurement of products of docosahexaenoic acid
peroxidation, neuroprostanes, and neurofurans. Methods Enzymol. 2007, 433, 127–143.
61. Song, W.L.; Lawson, J.A.; Reilly, D.; Rokach, J.; Chang, C.T.; Giasson, B.; FitzGerald, G.A.
Neurofurans, novel indices of oxidant stress derived from docosahexaenoic acid. J. Biol. Chem.
2008, 283, 6–16.
62. Signorini, C.; Ciccoli, L.; Leoncini, S.; Carloni, S.; Perrone, S.; Comporti, M.; Balduini, W.;
Buonocore, G. Free iron, total F-isoprostanes and total F-neuroprostanes in a model of neonatal
hypoxic-ischemic encephalopathy: Neuroprotective effect of melatonin. J. Pineal Res. 2009, 46,
148–154.
63. Balduini, W.; Carloni, S.; Perrone, S.; Bertrando, S.; Tataranno, M.L.; Negro, S.; Proietti, F.;
Longini, M.; Buonocore, G. The use of melatonin in hypoxic-ischemic brain damage: An
experimental study. J. Matern. Fetal. Neonatal Med. 2012, 25, 119–124.
64. Welin, A.K.; Svedin, P.; Lapatto, R.; Sultan, B.; Hagberg, H.; Gressens, P.; Kjellmer, I.;
Mallard, C. Melatonin reduces inflammation and cell death in white matter in the mid-gestation
fetal sheep following umbilical cord occlusion. Pediatr. Res. 2007, 61, 153–158.
65. Eskiocak, S.; Tutunculer, F.; Basaran, U.N.; Taskiran, A.; Cakir, E. The Effect of melatonin on
protein oxidation and nitric oxide in the brain tissue of hypoxic neonatal rats. Brain Dev. 2007,
29, 19–24.
66. Witko-Sarsat, V.; Friedlander, M.; Capeillere-Blandin, C.; Nguyen-Khoa, T.; Nguyen, A.T.;
Zingraff, J.; Jungers, P.; Descamps-Latscha, B. Advanced oxidation protein products as a novel
marker of oxidative stress in uremia. Kidney Int. 1996, 49, 1304–1313.
67. Andrabi, S.A.; Sayeed, I.; Siemen, D.; Wolf, G.; Horn, T.F. Direct Inhibition of the
mitochondrial permeability transition pore: A possible mechanism responsible for anti-apoptotic
effects of melatonin. FASEB J. 2004, 18, 869–871.
68. Wang, X.; Zhu, S.; Pei, Z.; Drozda, M.; Stavrovskaya, I.G.; Del Signore, S.J.; Cormier, K.;
Shimony, E.M.; Wang, H.; Ferrante, R.J. et al. Inhibitors of cytochrome c release with
therapeutic potential for huntington’s disease. J. Neurosci. 2008, 28, 9473–9485.
Int. J. Mol. Sci. 2013, 14 9391

69. Jou, M.J.; Peng, T.I.; Reiter, R.J.; Jou, S.B.; Wu, H.Y.; Wen, S.T. Visualization of the
antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced
apoptosis of rat brain astrocytes. J. Pineal Res. 2004, 37, 55–70.
70. Kilic, E.; Kilic, U.; Reiter, R.J.; Bassetti, C.L.; Hermann, D.M. Tissue-plasminogen
activator-induced ischemic brain injury is reversed by melatonin: Role of inos and akt. J. Pineal Res.
2005, 39, 151–155.
71. Jang, M.H.; Jung, S.B.; Lee, M.H.; Kim, C.J.; Oh, Y.T.; Kang, I.; Kim, J.; Kim, E.H. Melatonin
attenuates amyloid beta25–35-induced apoptosis in mouse microglial bv2 cells. Neurosci. Lett.
2005, 380, 26–31.
72. Ebadi, M.; Sharma, S.K.; Ghafourifar, P.; Brown-Borg, H.; El Refaey, H. Peroxynitrite in
the pathogenesis of parkinson’s disease and the neuroprotective role of metallothioneins.
Methods Enzymol. 2005, 396, 276–298.
73. Alvira, D.; Tajes, M.; Verdaguer, E.; Acuna-Castroviejo, D.; Folch, J.; Camins, A.; Pallas, M.
Inhibition of the cdk5/p25 fragment formation may explain the antiapoptotic effects of melatonin
in an experimental model of parkinson’s disease. J. Pineal Res. 2006, 40, 251–258.
74. Ling, X.; Zhang, L.M.; Lu, S.D.; Li, X.J.; Sun, F.Y. Protective effect of melatonin on injuried
cerebral neurons is associated with bcl-2 protein over-expression. Zhongguo Yao Li Xue Bao
1999, 20, 409–414.
75. Sun, F.Y.; Lin, X.; Mao, L.Z.; Ge, W.H.; Zhang, L.M.; Huang, Y.L.; Gu, J. Neuroprotection by
melatonin against ischemic neuronal injury associated with modulation of DNA damage and
repair in the rat following a transient cerebral ischemia. J. Pineal Res. 2002, 33, 48–56.
76. Chung, S.Y.; Han, S.H. Melatonin attenuates kainic acid-induced hippocampal neurodegeneration
and oxidative stress through microglial inhibition. J. Pineal Res. 2003, 34, 95–102.
77. Feng, Z.; Cheng, Y.; Zhang, J.T. Long-term effects of melatonin or 17 beta-estradiol on
improving spatial memory performance in cognitively impaired, ovariectomized adult rats.
J. Pineal Res. 2004, 37, 198–206.
78. Deng, Y.Q.; Xu, G.G.; Duan, P.; Zhang, Q.; Wang, J.Z. Effects of melatonin on
wortmannin-induced TAU hyperphosphorylation. Acta Pharmacol. Sin. 2005, 26, 519–526.
79. Rosenstein, R.E.; Cardinali, D.P. Central gabaergic mechanisms as targets for melatonin activity
in brain. Neurochem. Int. 1990, 17, 373–379.
80. Molina-Carballo, A.; Munoz-Hoyos, A.; Sanchez-Forte, M.; Uberos-Fernandez, J.;
Moreno-Madrid, F.; Acuna-Castroviejo, D. Melatonin increases following convulsive seizures
may be related to its anticonvulsant properties at physiological concentrations. Neuropediatrics
2007, 38, 122–125.
81. Prada, C.; Udin, S.B.; Wiechmann, A.F.; Zhdanova, I.V. Stimulation of melatonin receptors
decreases calcium levels in xenopus tectal cells by activating gaba(c) receptors. J. Neurophysiol.
2005, 94, 968–978.
82. Prada, C.; Udin, S.B. Melatonin decreases calcium levels in retinotectal axons of xenopus laevis
by indirect activation of group iii metabotropic glutamate receptors. Brain Res. 2005, 1053, 67–76.
83. Buonocore, G.; Perrone, S.; Bracci, R. Free radicals and brain damage in the newborn. Biol.
Neonate 2001, 79, 180–186.
Int. J. Mol. Sci. 2013, 14 9392

84. Blomgren, K.; Hagberg, H. Free radicals, mitochondria, and hypoxia-ischemia in the developing
brain. Free Radic. Biol. Med. 2006, 40, 388–397.
85. Kumar, A.; Mittal, R.; Khanna, H.D.; Basu, S. Free radical injury and blood-brain barrier
permeability in hypoxic-ischemic encephalopathy. Pediatrics 2008, 122, 722–727.
86. Alonso-Alconada, D.; Hilario, E.; Alvarez, F.J.; Alvarez, A. Apoptotic cell death correlates with
ros overproduction and early cytokine expression after hypoxia-ischemia in fetal lambs.
Reprod. Sci. 2012, 19, 754–763.
87. Watanabe, K.; Wakatsuki, A.; Shinohara, K.; Ikenoue, N.; Yokota, K.; Fukaya, T. Maternally
administered melatonin protects against ischemia and reperfusion-induced oxidative
mitochondrial damage in premature fetal rat brain. J. Pineal Res. 2004, 37, 276–280.
88. Hutton, L.C.; Abbass, M.; Dickinson, H.; Ireland, Z.; Walker, D.W. Neuroprotective properties
of melatonin in a model of birth asphyxia in the spiny mouse (Acomys cahirinus). Dev. Neurosci.
2009, 31, 437–451.
89. Fu, J.; Zhao, S.D.; Liu, H.J.; Yuan, Q.H.; Liu, S.M.; Zhang, Y.M.; Ling, E.A.; Hao, A.J.
Melatonin promotes proliferation and differentiation of neural stem cells subjected to hypoxia
in vitro. J. Pineal Res. 2011, 51, 104–112.
90. Kilic, U.; Kilic, E.; Reiter, R.J.; Bassetti, C.L.; Hermann, D.M. Signal transduction pathways
involved in melatonin-induced neuroprotection after focal cerebral ischemia in mice. J. Pineal
Res. 2005, 38, 67–71.
91. Koh, P.O. Melatonin prevents the injury-induced decline of akt/forkhead transcription factors
phosphorylation. J. Pineal Res. 2008, 45, 199–203.
92. Zhou, J.; Zhang, S.; Zhao, X.; Wei, T. Melatonin impairs nadph oxidase assembly and decreases
superoxide anion production in microglia exposed to amyloid-beta1–42. J. Pineal Res. 2008, 45,
157–165.
93. Koh, P.O. Melatonin prevents ischemic brain injury through activation of the mtor/p70s6 kinase
signaling pathway. Neurosci. Lett. 2008, 444, 74–78.
94. Fjaerli, O.; Lund, T.; Osterud, B. The effect of melatonin on cellular activation processes in
human blood. J. Pineal Res. 1999, 26, 50–55.
95. Baykal, A.; Iskit, A.B.; Hamaloglu, E.; Guc, M.O.; Hascelik, G.; Sayek, I. Melatonin modulates
mesenteric blood flow and TNF concentrations after lipopolysaccharide challenge. Eur. J. Surg.
2000, 166, 722–727.
96. Silva, S.O.; Rodrigues, M.R.; Ximenes, V.F.; Bueno-da-Silva, A.E.; Amarante-Mendes, G.P.;
Campa, A. Neutrophils as a specific target for melatonin and kynuramines: Effects on cytokine
release. J. Neuroimmunol. 2004, 156, 146–152.
97. Wang, H.; Wei, W.; Shen, Y.X.; Dong, C.; Zhang, L.L.; Wang, N.P.; Yue, L.; Xu, S.Y.
Protective effect of melatonin against liver injury in mice induced by bacillus calmette-guerin
plus lipopolysaccharide. World J. Gastroenterol. 2004, 10, 2690–2696.
98. Perianayagam, M.C.; Oxenkrug, G.F.; Jaber, B.L. Immune-modulating effects of melatonin,
N-acetylserotonin, and N-acetyldopamine. Ann. N.Y. Acad. Sci. 2005, 1053, 386–393.
99. Carrillo-Vico, A.; Lardone, P.J.; Fernandez-Santos, J.M.; Martin-Lacave, I.; Calvo, J.R.;
Karasek, M.; Guerrero, J.M. Human lymphocyte-synthesized melatonin is involved in the regulation
of the interleukin-2/interleukin-2 receptor system. J. Clin. Endocrinol. MeTable 2005, 90, 992–1000.
Int. J. Mol. Sci. 2013, 14 9393

100. Gitto, E.; Reiter, R.J.; Sabatino, G.; Buonocore, G.; Romeo, C.; Gitto, P.; Bugge, C.;
Trimarchi, G.; Barberi, I. Correlation among cytokines, bronchopulmonary dysplasia and
modality of ventilation in preterm newborns: Improvement with melatonin treatment. J. Pineal Res.
2005, 39, 287–293.
101. Steinhilber, D.; Brungs, M.; Werz, O.; Wiesenberg, I.; Danielsson, C.; Kahlen, J.P.; Nayeri, S.;
Schrader, M.; Carlberg, C. The nuclear receptor for melatonin represses 5-lipoxygenase gene
expression in human B lymphocytes. J. Biol. Chem. 1995, 270, 7037–7040.
102. Mayo, J.C.; Sainz, R.M.; Tan, D.X.; Hardeland, R.; Leon, J.; Rodriguez, C.; Reiter, R.J.
Anti-inflammatory actions of melatonin and its metabolites, N1-acetyl-N2-formyl-5-
methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in macrophages.
J. Neuroimmunol. 2005, 165, 139–149.
103. Deng, W.G.; Tang, S.T.; Tseng, H.P.; Wu, K.K. Melatonin suppresses macrophage
cyclooxygenase-2 and inducible nitric oxide synthase expression by inhibiting p52 acetylation
and binding. Blood 2006, 108, 518–524.
104. Cardinali, D.P.; Ritta, M.N.; Fuentes, A.M.; Gimeno, M.F.; Gimeno, A.L. Prostaglandin E
release by rat medial basal hypothalamus in vitro. Inhibition by melatonin at submicromolar
concentrations. Eur. J. Pharmacol. 1980, 67, 151–153.
105. Cardinali, D.P.; Ritta, M.N. The role of prostaglandins in neuroendocrine junctions: Studies in
the pineal gland and the hypothalamus. Neuroendocrinology 1983, 36, 152–160.
106. Carrillo-Vico, A.; Garcia-Maurino, S.; Calvo, J.R.; Guerrero, J.M. Melatonin counteracts the
inhibitory effect of PGE2 on IL-2 production in human lymphocytes via its mt1 membrane
receptor. FASEB J. 2003, 17, 755–757.
107. Bilici, D.; Akpinar, E.; Kiziltunc, A. Protective effect of melatonin in carrageenan-induced acute
local inflammation. Pharmacol. Res. 2002, 46, 133–139.
108. Acuna-Castroviejo, D.; Escames, G.; Lopez, L.C.; Hitos, A.B.; Leon, J. Melatonin and nitric
oxide: Two required antagonists for mitochondrial homeostasis. Endocrine 2005, 27, 159–168.
109. Leon, J.; Macias, M.; Escames, G.; Camacho, E.; Khaldy, H.; Martin, M.; Espinosa, A.;
Gallo, M.A.; Acuna-Castroviejo, D. Structure-related inhibition of calmodulin-dependent
neuronal nitric-oxide synthase activity by melatonin and synthetic kynurenines. Mol. Pharmacol.
2000, 58, 967–975.
110. Leon, J.; Escames, G.; Rodriguez, M.I.; Lopez, L.C.; Tapias, V.; Entrena, A.; Camacho, E.;
Carrion, M.D.; Gallo, M.A.; Espinosa, A. et al. Inhibition of neuronal nitric oxide synthase
activity by N1-acetyl-5-methoxykynuramine, a brain metabolite of melatonin. J. Neurochem.
2006, 98, 2023–2033.
111. Jimenez-Ortega, V.; Cano, P.; Cardinali, D.P.; Esquifino, A.I. 24-Hour variation in gene
expression of redox pathway enzymes in rat hypothalamus: Effect of melatonin treatment. Redox
Rep. 2009, 14, 132–138.
112. Tapias, V.; Escames, G.; Lopez, L.C.; Lopez, A.; Camacho, E.; Carrion, M.D.; Entrena, A.;
Gallo, M.A.; Espinosa, A.; Acuna-Castroviejo, D. Melatonin and its brain metabolite
N(1)-acetyl-5-methoxykynuramine prevent mitochondrial nitric oxide synthase induction in
parkinsonian mice. J. Neurosci. Res. 2009, 87, 3002–3010.
Int. J. Mol. Sci. 2013, 14 9394

113. Leon, J.; Vives, F.; Crespo, E.; Camacho, E.; Espinosa, A.; Gallo, M.A.; Escames, G.;
Acuna-Castroviejo, D. Modification of nitric oxide synthase activity and neuronal response in rat
striatum by melatonin and kynurenine derivatives. J. Neuroendocrinol. 1998, 10, 297–302.
114. Chandrasekaran, A.; Ponnambalam, G.; Kaur, C. Domoic acid-induced neurotoxicity in the
hippocampus of adult rats. Neurotox Res. 2004, 6, 105–117.
115. Escames, G.; Khaldy, H.; Leon, J.; Gonzalez, L.; Acuna-Castroviejo, D. Changes in iNOS
activity, oxidative stress and melatonin levels in hypertensive patients treated with lacidipine.
J. Hypertens. 2004, 22, 629–635.
116. Escames, G.; Acuna-Castroviejo, D.; Lopez, L.C.; Tan, D.X.; Maldonado, M.D.;
Sanchez-Hidalgo, M.; Leon, J.; Reiter, R.J. Pharmacological utility of melatonin in the treatment
of septic shock: Experimental and clinical evidence. J. Pharm. Pharmacol. 2006, 58, 1153–1165.
117. Escames, G.; Lopez, L.C.; Tapias, V.; Utrilla, P.; Reiter, R.J.; Hitos, A.B.; Leon, J.;
Rodriguez, M.I.; Acuna-Castroviejo, D. Melatonin counteracts inducible mitochondrial nitric
oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice. J. Pineal
Res. 2006, 40, 71–78.
118. Lopez, L.C.; Escames, G.; Tapias, V.; Utrilla, P.; Leon, J.; Acuna-Castroviejo, D. Identification
of an inducible nitric oxide synthase in diaphragm mitochondria from septic mice: Its relation
with mitochondrial dysfunction and prevention by melatonin. Int. J. Biochem. Cell Biol. 2006,
38, 267–278.
119. Srinivasan, V.; Pandi-Perumal, S.R.; Spence, D.W.; Kato, H.; Cardinali, D.P. Melatonin in septic
shock: Some recent concepts. J. Crit. Care 2010, 25, 656.e1–656.e6.
120. Lopez, L.C.; Escames, G.; Ortiz, F.; Ros, E.; Acuna-Castroviejo, D. Melatonin restores the
mitochondrial production of ATP in septic mice. Neuro Endocrinol. Lett. 2006, 27, 623–630.
121. Escames, G.; Lopez, L.C.; Ortiz, F.; Lopez, A.; Garcia, J.A.; Ros, E.; Acuna-Castroviejo, D.
Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice. FEBS J. 2007,
274, 2135–2147.
122. Pei, Z.; Cheung, R.T. Pretreatment with melatonin exerts anti-inflammatory effects against
ischemia/reperfusion injury in a rat middle cerebral artery occlusion stroke model. J. Pineal Res.
2004, 37, 85–91.
123. Lee, M.Y.; Kuan, Y.H.; Chen, H.Y.; Chen, T.Y.; Chen, S.T.; Huang, C.C.; Yang, I.P.; Hsu, Y.S.;
Wu, T.S.; Lee, E.J. Intravenous administration of melatonin reduces the intracerebral cellular
inflammatory response following transient focal cerebral ischemia in rats. J. Pineal Res. 2007,
42, 297–309.
124. Koh, P.O. Melatonin regulates nitric oxide synthase expression in ischemic brain injury. J. Vet.
Med. Sci. 2008, 70, 747–750.
125. Mohan, N.; Sadeghi, K.; Reiter, R.J.; Meltz, M.L. The neurohormone melatonin inhibits
cytokine, mitogen and ionizing radiation induced NF- B. Biochem. Mol. Biol. Int. 1995, 37,
1063–1070.
126. Reiter, R.J.; Calvo, J.R.; Karbownik, M.; Qi, W.; Tan, D.X. Melatonin and its relation to the
immune system and inflammation. Ann. N.Y. Acad. Sci. 2000, 917, 376–386.
127. Hardeland, R. Antioxidative protection by melatonin: Multiplicity of mechanisms from radical
detoxification to radical avoidance. Endocrine 2005, 27, 119–130.
Int. J. Mol. Sci. 2013, 14 9395

128. Kaur, C.; Sivakumar, V.; Lu, J.; Tang, F.R.; Ling, E.A. Melatonin attenuates hypoxia-induced
ultrastructural changes and increased vascular permeability in the developing hippocampus.
Brain Pathol. 2008, 18, 533–547.
129. Jan, J.E.; Wasdell, M.B.; Freeman, R.D.; Bax, M. Evidence supporting the use of melatonin in
short gestation infants. J. Pineal Res. 2007, 42, 22–27.
130. Gitto, E.; Reiter, R.J.; Cordaro, S.P.; La Rosa, M.; Chiurazzi, P.; Trimarchi, G.; Gitto, P.;
Calabro, M.P.; Barberi, I. Oxidative and inflammatory parameters in respiratory distress
syndrome of preterm newborns: Beneficial effects of melatonin. Am. J. Perinatol. 2004, 21,
209–216.
131. Gitto, E.; Reiter, R.J.; Amodio, A.; Romeo, C.; Cuzzocrea, E.; Sabatino, G.; Buonocore, G.;
Cordaro, V.; Trimarchi, G.; Barberi, I. Early indicators of chronic lung disease in preterm infants
with respiratory distress syndrome and their inhibition by melatonin. J. Pineal Res. 2004, 36,
250–255.
132. Buonocore, G.; Groenendaal, F. Anti-oxidant strategies. Semin. Fetal. Neonatal Med. 2007, 12,
287–295.
133. Robertson, N.J.; Faulkner, S.; Fleiss, B.; Bainbridge, A.; Andorka, C.; Price, D.; Powell, E.;
Lecky-Thompson, L.; Thei, L.; Chandrasekaran, M. et al. Melatonin augments hypothermic
neuroprotection in a perinatal asphyxia model. Brain 2013, 136, 90–105.
134. Waldhauser, F.; Waldhauser, M.; Lieberman, H.R.; Deng, M.H.; Lynch, H.J.; Wurtman, R.J.
Bioavailability of oral melatonin in humans. Neuroendocrinology 1984, 39, 307–313.
135. Aldhous, M.; Franey, C.; Wright, J.; Arendt, J. Plasma concentrations of melatonin in man
following oral absorption of different preparations. Br. J. Clin. Pharmacol. 1985, 19, 517–521.
136. Lane, E.A.; Moss, H.B. Pharmacokinetics of melatonin in man: First pass hepatic metabolism.
J. Clin. Endocrinol. MeTable 1985, 61, 1214–1216.
137. Merchant, N.M.; Azzopardi, D.V.; Hawwa, A.F.; McElnay, J.C.; Middleton, B.; Arendt, J.;
Arichi, T.; Gressens, P.; Edwards, A.D. Pharmacokinetics of Melatonin in Preterm Infants. Br. J.
Clin. Pharmacol. 2013, doi:10.1111/bcp.12092.

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).