HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Reiter, R. J. Articles by Kilic, E. Search for Related Content PubMed PubMed Citation Articles by Reiter, R. J. Articles by Kilic, E.

---

MINIREVIEW

When Melatonin Gets on Your Nerves: Its Beneficial Actions in Experimental Models of Stroke

Russel J. Reiter^*,1, Dun-xian Tan^*, Josefa Leon^*, Ülkan Kilic and Ertugrul Kilic

^* Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78229; and Department of Neurology, University Hospital of Zürich, Zürich, Switzerland

¹ To whom requests for reprints should be addressed at Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, MC 7762, San Antonio, TX 78229-3900. E-mail: reiter{at}uthscsa.edu

	Abstract

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

 
This article summarizes the evidence that endogenously produced and exogenously administered melatonin reduces the degree of tissue damage and limits the biobehavioral deficits associated with experimental models of ischemia/reperfusion injury in the brain (i.e., stroke). Melatonin’s efficacy in curtailing neural damage under conditions of transitory interruption of the blood supply to the brain has been documented in models of both focal and global ischemia. In these studies many indices have been shown to be improved as a consequence of melatonin treatment. For example, when given at the time of ischemia or reperfusion onset, melatonin reduces neurophysiological deficits, infarct volume, the degree of neural edema, lipid peroxidation, protein carbonyls, DNA damage, neuron and glial loss, and death of the animals. Melatonin’s protective actions against these adverse changes are believed to stem from its direct free radical scavenging and indirect antioxidant activities, possibly from its ability to limit free radical generation at the mitochondrial level and because of yet-undefined functions. Considering its high efficacy in overcoming much of the damage associated with ischemia/reperfusion injury, not only in the brain but in other organs as well, its use in clinical trials for the purpose of improving stroke outcome should be seriously considered.

Key Words: ischemia/reperfusion injury • stroke • brain oxidative stress • antioxidant • melatonin • free radicals

	Introduction

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

 
Stroke—also referred to as ischemia/reperfusion, a cerebrovascular accident, or brain attack—often has devastating neuropathological, neurophysiological, and biobehavioral consequences that commonly result in permanent disability or death. Certain individuals are at increased risk for neurological stroke; this group includes individuals with hypertension, atherosclerosis, heart disease and other cardiovascular disorders, and those who smoke cigarettes (1–3). Also, obesity, advanced age, and diabetes mellitus are well-known risk factors for stroke.

In the context of this review, the term "hypoxia/anoxia" is interchangeable with the term "ischemia." There are essentially two types of ischemia that occur in the brain: global ischemia and focal ischemia (4). Global ischemia occurs when the oxygen (O₂) to supply to the entire brain is interrupted or severely diminished; this occurs, for example, during cardiac arrest, asphyxia, or carbon monoxide poisoning. Normally, in humans cerebral blood flow is on the order of 50 ml/100 g brain/min. When flow is reduced to less than 18 ml/100 g brain/min, neurological injury results (5). Given the high requirement of the central nervous system (CNS) for O₂, the oxygenated blood supply to the brain must be reestablished within minutes to prevent severe neurological damage. Unfortunately, reperfusion and reoxygenation of O₂-deprived tissue also causes extensive molecular destruction resulting in neurological deficits.

Global ischemia for brief periods differentially damages neurons. In particular, resuscitation of individuals after a prolonged interval of cardiac arrest (several minutes), typically leads to extensive damage to the pyramidal neurons of the CA1 region of the hippocampus. Somewhat less easily damaged neurons are found in layers 3, 5, and 6 of the cerebral cortex and neurons in the striatum, hypothalamus, and cerebellum (6, 7). By contrast, many neurons seem to be capable of tolerating 30 mins or more of ischemia/anoxia. Even when neurons are damaged, death of these cells typically does not occur until 1–5 days following reperfusion/reoxygenation. When neural ischemia (global or local) is prolonged by 60 mins or more, most cells in the ischemic area die and an infarct develops.

Local (focal) ischemia occurs when the blood supply to a portion of the brain is interrupted, such as when a blood vessel ruptures (hemorrhagic stroke) or when a thrombus (clot), often in an atherosclerotic vessel, occurs. Hemorrhagic stroke is further complicated by the bleeding that percolates into the brain since hemoglobin is neurotoxic. It is not uncommon that rupture of a blood vessel in the brain leads to cerebral vasospasm and secondary ischemia. During local ischemia, the neurons and glia nearest the center of ischemic zone, sometimes referred to as the ischemic core or umbra, suffers the greatest amount of damage, while the surrounding area, the ischemic penumbra, which receives some oxygenated blood, becomes relatively less hypoxic and therefore shows less extensive damage.

Stroke is the third most frequent cause of medically related deaths in North America and Europe and the second leading cause of morbidity. Treatments or actions that reduce the incidence of stroke include the use of drugs to reduce hypertension and consuming diets rich in fruits and vegetables that are high in antioxidants; a high-antioxidant diet seems to reduce the incidence of atherosclerosis, thereby ameliorating the likelihood of developing both a hemorrhagic or a thrombotic ischemic event.

The neural damage that results during ischemia is a consequence of a variety of negative factors. To prevent extensive damage, it is most important to restore the blood supply to the ischemic tissue as quickly as possible; however, in doing so, reperfusion with oxygenated blood causes further molecular destruction and neuronal/glial loss. Factors generally believed to be culpable for ischemia/reperfusion (I/R) injury include a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The oxygen-derived reactants of particular interest include the superoxide anion radical (O₂^•–), hydrogen peroxide (H₂O₂), and the highly toxic hydroxyl radical (•OH). The most damaging RNS include nitric oxide (NO•) and especially the peroxynitrite anion (ONOO^–; Fig. 1; Refs. 8, 9).

View larger version (32K): [in this window] [in a new window]   Figure 1. The processes associated with neural damage during ischemia/reperfusion injury are highly complex as illustrated in this figure. Of note is that elevated free radicals and related reactive oxygen species (ROS)/reactive nitrogen species (RNS) are documented culpable agents in this damage. Additionally, tissue damage also results from the extensive edema as well as from the activation of endothelial adhesion molecules that result in leukocyte infiltration into the damaged tissue. Another result of the cellular damage resulting from ischemia/reperfusion injury is a drop in ATP levels that contributes to additional cell death. In the current review, the ability of melatonin to scavenge ROS/RNS as well as to limit edema and reduce adhesion molecules is discussed. Each of these actions of melatonin contribute to its ability to reduce the severity of stroke. XO, xanthine oxidase; NOS, nitric oxide synthase; SOD, superoxide dismutase; MPO, myloperoxidase; TBARS, thiobarbituric acid reactive substances (lipid peroxidation products); 8OHdG, 8-hydroxy-2-dexoxyguanosine (damaged DNA product).

As already alluded to, the brain is particularly susceptible to damage by processes that involve free radicals. There are several reasons for this:

1. O₂ consumption by the brain is much greater than that in other tissues. Although only roughly 2% of total body weight, the brain utilizes 20% of the inhaled O₂. Thus, proportionately, the brain generates many more bellicose ROS than any other tissue.
   
2. The brain is rich in polyunsaturated fatty acids (PUFA), which are easily oxidized by toxic reactants. This, coupled with elevated radical generation, makes the brain highly vulnerable to damage.
   
3. The brain contains elevated amounts of non-heme iron. In the event iron becomes liberated and encounters H₂O₂, it generates, via the Fenton reaction, the highly reactive and destructive •OH.
   
4. In addition to iron, certain areas of the brain are rich in ascorbic acid (vitamin C). In the presence of free iron, vitamin C becomes a potent pro-oxidant.
   
5. Finally, the CNS has a relatively inefficient enzymatic antioxidative defense system. Given these features, it is not totally surprising that, during a lifetime, the CNS suffers more than its share of oxidative mutilation, be it due to stroke or other age-related diseases.
   

The current review summarizes a series of studies that have used the newly discovered direct free radical scavenger (15–20) and indirect antioxidant (21–24) melatonin to combat the morphological, physiological, biochemical, and molecular damage that is a result of I/R injury in the CNS. The extensive data relating to the specific mechanisms by which melatonin reduces tissue damage that is a consequence of interruption and reestablishment of the blood supply to an organ are beyond the scope of the current review, and the reader is directed elsewhere for this information (15, 19, 20, 25). Suffice it to state, melatonin’s ability to reduce molecular damage during I/R likely involves the receptor-independent direct detoxification of ROS/RNS (15, 19, 20) as well as receptor-mediated actions such as the modulation of anti-oxidative enzymes (21–24). Beyond these presumably primary protective actions, melatonin may well have other beneficial effects, such as limiting electron leakage from the electron transport chain (ETC) in the inner mitochondrial membrane (22); by reducing electron leakage from the ETC free radical, generation would likewise be lessened.

Of importance is that the high efficacy of melatonin in reducing the structural and functional deficits associated with transitory interruption and reestablishment of the blood supply is not uniquely confined to the CNS. Indeed, melatonin’s ability to limit I/R damage has been documented in many organs, such as heart (26), liver (27, 28), gastrointestinal tract (29), lung (30), eye (31, 32), kidney (33), and others.

	Melatonin: Relation to the Brain

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

 
While melatonin is produced in the CNS, specifically in the pineal gland (34), its production is by no means limited to this organ. Melatonin synthesis has also been documented to occur in the retinas (35), gastrointestinal tract (36, 37), some bone marrow cells (38, 39), peripheral lymphocytes (40), skin (41), and possibly in many other cells as well (42–44). At least in the pineal gland and retina, melatonin production is light:dark dependent with highest levels of the indoleamine being generated during the dark phase of the light:dark cycle. Besides its endogenous production, it is also ingested in the diet since melatonin is present in plants including edible foodstuffs (45, 46). Consumption of foodstuffs containing melatonin is followed by its absorption into the blood (45).

Although its synthesis occurs in a number of cells, the release of melatonin, at least in large amounts, seems to be restricted to the pineal gland. Certainly, surgical removal of the pineal gland eliminates the nocturnal increase in blood melatonin concentrations (34). The mechanisms of melatonin release remain enigmatic, although it is usually assumed that it is a consequence of simple diffusion of the indoleamine out of the pinealocytes. While a similar secretory process for melatonin may exist in other cells where it is produced, in these tissues its actions are confined to the immediate vicinity of its cells of production, and significant amounts typically do not escape into the systemic circulation. In these organs, melatonin has autocrine, intracrine, and pararine actions (40, 47).

In reference to the pineal gland, it has long been suspected that, in addition to its discharge into the rich capillary plexus in the pineal gland, melatonin may also be released directly into the cerebrospinal fluid (CSF) of the third ventricle. Certainly, there are a variety of morphological characteristics of the structures in the vicinity of the pineal gland that would make secretion via this route a possibility (48). Recent studies in the sheep support the release of melatonin from the pineal gland directly into the third ventricle (49, 50). In a series of elegant experiments, this group convincingly showed that CSF melatonin concentrations are diminished when the pineal recess of the third ventricle of the sheep is obstructed; these data strongly imply that melatonin, released from the pineal gland, enters the CSF via the recess that invaginates the pineal gland in the posterodorsal aspect of the third ventricle of the sheep (50). If this ventricular route of secretion if further validated in the sheep, it would seem unusual if it were a unique feature of this species. The human pineal, as in the sheep, sits in a subcallosal position and possesses a pineal recess and other morphological modifications that would certainly allow the escape of pineal-derived melatonin directly into the third ventricle. The other important observation that Skinner and Malpaux (49) made was that nocturnal third ventricular CSF melatonin levels are several orders of magnitude higher than simultaneous concentrations measured in the peripheral blood (Fig. 2). This finding by itself implies a direct release of melatonin into the third ventricle or a very active mechanism for the transfer of melatonin from the blood through the choroid plexus and into the CSF. This latter possibly seems unlikely considering the reported data (50).

View larger version (19K): [in this window] [in a new window]   Figure 2. Concurrent melatonin levels in the plasma and the cerebrospinal fluid (CSF) of a sheep over a 24-hr light:dark cycle. As with all mammals, blood melatonin concentrations in the sheep rise from a value of <10 pg/ml during the day to 50–150 pg/ml at night; in contrast, the nocturnal rise in CSF melatonin values are orders of magnitude higher than in simultaneous plasma samples. Likewise, it was shown that in the sheep, melatonin is released from the pineal gland directly into third ventricular CSF. This being the case, the brain is seemingly exposed to much higher concentrations of melatonin at night than are other organs. This is the only species in which a direct release of melatonin into the third ventricle has been convincingly demonstrated, but it is unlikely that it is the only species in which it occurs. From Skinner and Malpaux (49).

The observation of much higher melatonin levels in the CSF than in the blood points out another important feature concerning melatonin; that is, it is not in equilibrium in the body. This has actually been apparent for at least a decade but not widely recognized. In addition to the third ventricular fluid, several other fluids likewise contain concentrations of melatonin that exceed those in the blood when measured at the same time. Like the CSF, bile has levels of melatonin that are orders of magnitude higher than those in the blood (51); similarly, although not equivalent to those in the CSF or bile, ovarian follicular fluid (52) contains melatonin at concentrations that surpass those in the plasma. Likewise, cells that produce melatonin—and there seem to be more than originally anticipated—likely contain high concentrations of the indoleamine. Also, very high intracellular melatonin levels are generally compatible with optimal cellular function as illustrated by millimolar levels (compared to picomolar or low-nanomolar concentrations in mammalian blood) of this indoleamine in the unicell Gonyaulax polyedra (53). Furthermore, by the standard of blood levels, the yeast Saccharomyces cerevisiae has melatonin concentrations that go well beyond those considered the norm (54). Within the mammalian cell as well, when melatonin was measured in mitochondria of rat liver, they were preliminarily reported to be in the micromolar range (55). Nuclear levels of melatonin in mammalian cells also seem to exceed those in the blood (56). Thus, care must be exercised when defining the physiological concentration of melatonin. Clearly, melatonin concentrations differ among subcellular organelles and bodily fluids as well as between species. Hence, when the phrase "physiological level" is used to describe a particular melatonin concentration, it should be qualified on the basis of a specific organelle, a fluid, and a particular species (57).

For any antioxidant to protect neurons and glia from highly reactive free radicals and associated species, it is imperative that it gets into the brain in sufficient concentrations to do so. In this context, melatonin seems to have an advantage over some antioxidants that are not normally generated in the brain. Vitamins E and C, for example, are not particularly effective in crossing the blood-brain barrier. Conversely, within 10 mins after its peripheral administration, melatonin is already detected in the CNS (56–58).

In addition to its ability to enter the brain, it is important that an antioxidant have wide intracellular distribution and multiple actions to be maximally effective in protecting against oxidative stress. Here again, melatonin may be superior to some other classic antioxidants. Within neurons and glia, vitamin E is restricted, because of its solubility, to lipid-rich cellular membranes; conversely, the highest concentrations of vitamin C are found in the cytosol. Melatonin’s intracellular localization seems to be much more diffused, although the data are still incomplete. Melatonin’s high lipophilicity would predict its presence in cellular membranes. However, immunocytochemical and radioimmunoassay data have shown that it is also present in mitochondria (55) and nuclei (58) of cells. Certainly, melatonin has repeatedly been shown to prevent free radical damage to membrane lipids (59–61), to cytosolic proteins (62–64), and to nuclear (65–67) and mitochondrial (68, 69) DNA. Melatonin’s wide intracellular distribution seems to relate to its amphilicity, the basis of which may be its O-methyl and N-acetyl residues (70). Considering the extremely short distances that most highly reactive radicals travel before mutilating a bystander molecule, melatonin (or any free radical scavenger) must be on-site when a radical is produced. In a number of in vivo comparative studies, melatonin has been shown to be a more potent protector against free radical––mediated molecular destruction than either vitamin E or vitamin C (66, 71–73).

	Use of Antioxidants to Reduce the Consequences of Stroke

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

 
As illustrated in Figure 1, ROS and RNS appear to play a central role in the neuronal damage that occurs as a consequence of I/R injury (stroke) in the CNS. Annually, thousands of individuals die or are seriously incapacitated because of a transitory interruption of the blood supply to portions of the brain.

A number of therapeutic interventions have been tested in a variety of animals in an attempt to reduce the neuronal loss and neurophysiologic deficits associated with experimental stroke. Some of these experimental treatments include reducing body temperature, giving inhibitors of NO• synthesis, injecting Ca²⁺ channel blockers, providing agents that reduce the binding of glutamate to its NMDA receptor, and providing either enzymatic or nonenzymatic antioxidants. The intent of the use of each of these treatments is to limit free radical damage in the affected brain region. Some of the naturally occurring and synthetic antioxidants that have been used include superoxide dismutase (SOD or SOD mimics), metal chelators such as desferrioxamine, Ebselen (glutathione peroxidase mimic), -lipoic acid, vitamins E and C as well as derivatives of these molecules, and lazaroids (e.g., tirilazad mesylate or U-74006F). Additionally, the spin-trapping agent phenyl-tertbutylnitrone (PBN) has been examined as to its efficacy in reducing molecular and cellular damage in the brain due to I/R injury. The perceived success of these treatments is dependent on the specific animal model in which the agent was tested, the duration of the ischemic insult, the length of follow-up after reperfusion was established, the basal levels of endogenous brain antioxidants at the time I/R occurred, and other factors. The intent of each of these treatments was primarily to reduce the free radical damage associated with transient interruption of the blood flow to the brain followed by its reperfusion with oxygenated blood.

Melatonin has also been widely tested as to its ability to reduce molecular and cellular damage in the CNS during I/R injury. The actual number of studies that have been performed using melatonin in its context of an inhibitor of I/R injury is surprisingly extensive considering this indoleamine has been known to function as an antioxidant for only roughly a decade (74). Additionally, several other summaries of this subject have appeared elsewhere and may be consulted for additional details and perspectives (75–78).

As will be apparent in the subsequent paragraphs, melatonin’s effects in studies of experimental I/R injury have been unexpectedly successful. The reasons for this are not immediately apparent but presumably relate to its broad-spectrum functions as an antioxidant (19) and to the fact that not only is melatonin highly effective as a direct and indirect antioxidant (20–22), but some of the products that are formed when melatonin interacts with ROS are also highly effective in preventing oxidative damage. For example, N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and N¹-acetyl-5-methoxykynuramine (AMK), both of which are metabolites of melatonin (15, 79–82), are highly efficient radical scavengers (83–86).

The initial study examining melatonin’s ability to reduce tissue damage during a transient interruption of the blood supply to the brain followed rather soon after the discoveries that documented the antioxidant properties of melatonin (15, 16, 74, 87). Interestingly, in this report the authors did not determine whether exogenously administered pharmacological levels of melatonin would abate the degree of I/R injury but rather tested the efficacy of depressed endogenous melatonin levels (due to surgical removal of the pineal gland) on neural damage following the temporary interruption of the blood supply to the brain. This would have to be considered a high-risk study in terms of a positive outcome considering that pinealectomy removes only one source of melatonin so that animals, in this case rats (88), were not totally depleted of endogenous melatonin levels, although they were deficient with regard to circulating melatonin concentrations. Despite this, the rats that were deficient in melatonin had larger infarct volumes, more terminal transferase biotinylated-dUTP nick end label (TUNEL)–positive neurons, and fewer surviving cells (Nissl stain) in the brain 4 or 6 hrs after temporary bilateral occlusion of both common carotid arteries (88). The TUNEL assay identifies cells dying because of apoptosis or necrosis after DNA damage. This group quickly followed their initial report with a second study that confirmed that a relative melatonin deficiency is associated with an exaggerated infarct volume and more extensive cellular DNA damage in the brain after a period of transitory focal ischemia followed by reperfusion with oxygenated blood (89). This group also provided data showing that exogenously administered pharmacological levels of melatonin (4 x 2.5 mg/kg body weight [BW] before ischemia onset) reduced the amount of tissue loss in the CNS after I/R.

The ability of endogenous levels of melatonin to contribute protection to the brain during I/R in rats was also confirmed by Kilic and colleagues (31). In this study, both pinealectomized and pineal intact rats were subjected to a reversible 120-min focal brain ischemia. As a consequence of the relative melatonin deficiency that followed pine-alectomy, infarct volume after endovascular middle cerebral artery occlusion (MCAO) was exaggerated relative to that in the intact animals, and supplemental melatonin (4 mg/kg BW before ischemia onset and before reperfusion) significantly limited brain damage 22 hrs after reperfusion onset. This report also showed that the neurological deficits associated with I/R injury in pinealectomized rats were ameliorated by giving melatonin exogenously.

The results showing that attenuated melatonin levels enhance neurological damage after an I/R episode have noteworthy implications given that melatonin production and its secretion from the pineal gland diminishes with age (90–92). Hence, considering that older humans have lower endogenous melatonin concentrations than their young counterparts, when they experience a stroke it would be expected that the degree of neural damage would be enhanced in an older population. Certainly, Manev and Uz (93) suggest this in an opinion paper, and an obvious implication is that aged individuals who have reduced melatonin levels and who are prone to stroke may benefit from melatonin supplementation.

Other investigations have also examined melatonin’s ability to reduce neurological damage after focal I/R of the brain. For example, Cho and colleagues (94) specifically investigated the consequences of I/R on the CA1 pyramidal neurons of the hippocampus of rats after 10, 20, or 30 mins of ischemia followed by reperfusion for 7 days. The hippocampal pyramidal neurons are frequently lost during stroke since the neurotransmitter glutamate, which is abundant at synapses that end on pyramidal neurons, is massively released during brain injury. The consequence of the release of glutamate is excitotoxicity, a process that is highly damaging to postsynaptic neurons including the pyramidal cells of the hippocampus. Excitotoxicity is deadly to neurons because it ultimately leads to pronounced free radical generation, molecular damage, and cell death. Cho et al. (94) found that melatonin (10 mg/kg BW) protected the pyramidal cells from excitotoxicity-induced cell death that followed the induction of experimental stroke. Indeed, in other models of excitotoxicity, melatonin has also proven protective of neurons against glutamate-induced damage (95–97). These observations are of considerable importance given that excitotoxicity-mediated neuronal death, besides being involved in I/R injury, is believed to be a factor in age-related neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases as well (98–100).

While the previously summarized findings certainly indicate that one of the mechanisms by which melatonin reduces neural damage after I/R involved its ability neutralize free radicals, actual documentation of this had to await a study by Li et al. (101). This group placed microdialysis probes into the ischemic area of the brain of rats and used salicylate trapping to estimate •OH generation in either the presence or the absence of supplemental melatonin administration. In this study, 2,3-dihydroxybenzoic acid (DHBA), a product that is generated when salicylate scavenges an •OH, was found to be elevated in microdialysates recovered from the rat brain during ischemia and reperfusion. These elevated values were significantly reduced when melatonin was given before the dialysates were collected from the brain. In this situation, DHBA levels were lowered because melatonin, more effectively than salicylate, scavenged the newly formed •OH. These findings certainly support the conclusion that melatonin’s efficacy in limiting neuronal death and infarct volume that is a consequence of MCAO may well relate to the ability of the indole to directly detoxify the devastatingly toxic •OH. Beyond this action of melatonin, however, it is likely that the indole’s protective effects in the brain also involve preservation of mitochondrial physiology (22, 23, 102), stimulation of antioxidative enzymes (24), as well as other actions (76).

Apoptosis is a major process by which cells die in the CNS as well as elsewhere after they are damaged by oxidative stress. Ling and colleagues (103) examined the effects of melatonin on the protooncogenes, bcl-2 and bax, in the rat brain during hypoxia and reoxygenation associated with MCAO. The bcl-2, which enhances cell survival, was upregulated in the penumbral area of an infarct in the rat brain after MCAO when melatonin was given to protect against free radical damage; bax levels, however, were not altered by either a period of ischemia follow by reperfusion or by exogenous melatonin administration. Since the bcl-2 levels were elevated as a result of melatonin treatment, the bcl-2-to-bax ratio was likewise increased. These results are consistent with melatonin’s ability to reduce cellular loss in the CNS by inhibition of apoptosis, a process often found to be reduced as a consequence of the actions of melatonin in the brain (104, 105) and in other organs (106).

Sinha et al. (107) used another approach to estimate how effectively melatonin protected against infarct volume after induced hypoxia and reoxygenation of the brain. In this report, the infarct volume in the rat brain after a 2-hr occlusion of the middle cerebral artery followed by a 30-min internal of reperfusion was evaluated using magnetic resonance imaging (MRI). In the animals that received melatonin, the lesion size was estimated to be reduced by 50% compared to that in diluenttreated controls. Furthermore, using a number of behavioral tests, they established that the neurological deficits associated with MCAO were also less conspicuous in melatonin-treated rats. The presumptive mechanisms for the protective effects of melatonin in this study, as expressed by the authors (107), related to the ability to the indole to directly scavenge free radicals as well as its propensity to promote the activities of several antioxidative enzymes.

Kondoh et al. (108) and Torii and colleagues (109) also employed MRI signals to assess the amount of damaged tissue in the brain of rats subjected to MCAO (using an intraluminal occlusion technique) with and without concurrent melatonin administration. In this case, melatonin was given orally at doses of 6 mg/kg BW; the initial dose was administered immediately before onset of a 1-hr MCOA, while the second dose was given 24 hrs later. When the brain of rats was visualized, the T2-weighted signals, indicative of the water content of the brain, showed massive edema in the area of the brain that had been subjected to I/R. Oral melatonin treatment reduced the amount of edema by more than 50% with this reduction being greater in the cerebral cortex (59.8%) than in the striatum (34.2%). When the volume of edema was estimated in successive coronal brain slices, melatonin more effectively reduced cerebral cortical edema posterior as opposed to anterior to the bregma (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Figure 3. Reduction in cerebral edema in the rat brain after induced ischemia/reperfusion injury with and without melatonin treatment; the volume of edema was estimated using successive, from front to back, T2-weighted magnetic resonance images. Melatonin clearly limited the volume of edema at all cortical levels with the effect being somewhat greater caudal to the bregma. In these brains, the infarct volume correlated with the volume of edema; thus, melatonin treatment reduced not only edema volume but the size of the infarct as well. *P < 0.05 and **P < 0.001 compared with saline treated controls. The horizontal bar signifies a P < 0.01 between melatonin-and saline-treated rats over the length of the bar. From Torii et al. (109).

Intraarterial thread occlusion of the middle cerebral artery and its withdrawal after 30 mins was used by Kilic et al. (113) to induce I/R injury in mice. Also, in the study melatonin was given with the thrombolytic agent, recombinant tissue-plasminogen activator (t-PA), which is commonly used in humans to dissolve clots within blood vessels. This combination treatment was employed since it may become relevant given that humans with obstruction of a blood vessel supplying the brain are frequently given t-PA for thrombolysis. To test whether there are protective effects of melatonin in this model of I/R, Kilic and co-workers (113) also monitored cerebral flow using laser Doppler (LDF). Melatonin by itself caused a significant reduction in LDF, but when combined with t-PA, it was ineffective in altering blood flow to the cerebral cortex. At 24 hrs postischemic onset, melatonin-treated mice had significantly increased neuronal survival and diminished disseminate cell injury as assessed by cresyl violet and TUNEL staining. Melatonin, when combined with t-PA treatment, was also protective against cell death, although t-PA by itself also provided some neural protection. The protective actions of melatonin were associated with inhibition of caspase 3, an apoptosis-related enzyme. The authors concluded that the brain protection by melatonin in this I/R model was independent of any hemodynamic changes but did involve the inhibition of caspase 3, which was likely a result of melatonin-scavenging radicals at the mitochondrial level and the subsequent diminished release of cytochrome c from mitochondria (105).

A commonly used species to investigate the neural consequences of extensive neural hypoxia and reperfusion is the Mongolian gerbil (Meriones unguiculatus). The utility of this species in I/R studies stems from the incomplete circle of Willis at the base of the diencephalon. Because of this, when both common carotid or vertebral arteries are obstructed, ischemia is complete in the distribution area of these blood vessels since there is no collateral circulation to supply the region deprived of blood. Two studies that tested melatonin as a protective agent in the CNS have used this species (114, 115). In the first of these studies, bilateral occlusion using vascular clamps of both common carotid arteries followed by reperfusion of the ischemic tissue was followed by increases in cerebrocortical levels of nitric oxide (NO; as estimated by nitrite/nitrate concentrations) and rises in the second messenger, cyclic guanosine monophosphate (cGMP). It is believed that NO is in part responsible for N-methyl-D-aspartate glutamergic toxicity associated with ischemia. Melatonin, given in advance of ischemia, prevented the increases in both NO and cGMP in the frontal cerebral and cerebellar cortices (115). When it couples with O₂^{• –}, NO forms the highly toxic ONOO^–; this latter molecule is scavenged by melatonin (18–20), and this is one action that was assumed to explain the protective effects of melatonin in this I/R model.

The second study that used the Mongolian gerbil (114) measured more endpoints than did the investigation of Guerrero and coworkers (115). Again, bilateral occlusion of the common carotid arteries was the method employed to induce hypoxia of the forebrain. Melatonin (10 mg/kg BW) was given 30 mins before reperfusion onset and also at 1, 2, and 6 hrs postreperfusion. Cuzzocrea et al. (114) measured nitrite/nitrate levels, malondialdehyde concentrations, myloperoxidase activity, neural edema, locomotor activity, nitrotyrosine, and poly (ADP ribose) synthase staining of the hippocampal pyramidal neurons, pyramidal cell counts, and survival of the animals after I/R injury with and without melatonin treatment. For every parameter measured, a significant improvement was seen when the animals had been treated with melatonin (as opposed to diluent). Particularly noteworthy are the survival studies; 5 of 10 diluent-treated, I/R gerbils survived to 24 hrs, while 3 of 10 were alive at 48 hrs; conversely, 10 of 10 and 8 of 10 gerbils survived to 24 and 48 hrs, respectively, when they had been given melatonin.

Perhaps the most extensive series of investigations on the protective actions of melatonin against I/R damage to the rodent brain is that of Cheung and co-workers (77). In two recently published reports where the melatonin injection scheme was simplified (relative to that of the earlier studies), this group (116, 117) documented that a single intra-peritoneal injection of melatonin (5–15 mg/kg BW) before 3-hr endovascular MCAO has pronounced cerebroprotective actions. In these studies, infarct volume was reduced by roughly 40%, and an effect of melatonin via potential hemodynamic changes was ruled out since these parameters remained unchanged following administration of the indole. Even when a 5-mg/kg BW dose of melatonin commenced within 1 hr of ischemia onset (total duration of ischemia was 3 hrs), the protective actions of the indole were obvious and even more prominent when a second and third dose of melatonin were given at 21 and 45 hrs after reperfusion onset (118). As other investigators had shown, the I/R-induced rises in brain NO concentrations were also reduced in the melatonin-treated rats (119).

Compared to the number of focal I/R models that have been employed to test the cerebroprotective actions of melatonin, many fewer global I/R models have been used. The first of these was conducted by Wakatsuki and coworkers (120). Global I/R was induced in fetal rats by clamping the utero-ovarian arteries bilaterally for 20 mins and then reestablishing the blood flow to the pregnant uterus. A 10-mg/kg-BW melatonin dose was given intraperitoneally to the pregnant dam 1 hr in advance of uteroovarian artery occlusion. Following I/R, the fetal rat brain had elevated levels of products of lipid peroxidation and damaged DNA, with both changes being attenuated by the single melatonin injection. This study emphasizes another point that was previously established, that is, that melatonin readily passes through the placenta (121) and does so in amounts sufficient to protect the fetus from oxidative damage. The ability of melatonin to enter the fetus when administered to the mother has clear implications for its use in protecting the unborn fetus from free radical damage due to a variety agents.

The other model of global I/R that was used to test melatonin’s protective actions in the brain was cardiopulmonary arrest. In this case, cats were the experimental animals, and the duration of blood deprivation to the brain was 15 mins (122). This interruption of blood flow led to extensive loss of CA1 and CA4 hippocampal pyramidal neurons 8 days later, while neurological deficits were also obvious when measured on Days 1–7 post-ischemia. Continuously infused melatonin at a dose of 10 mg/kg BW/hr for 6 hrs commencing at 30 mins after reperfusion onset significantly reduced hippocampal neuronal loss and reduced the severity of the neurobehavioral deficits.

Collectively, the results of the studies summarized here are unambiguous in terms of the ability of melatonin to restrict CNS damage, which is a consequence of I/R. In each of these reports, melatonin administration was usually initiated essentially at the time of ischemia or reperfusion onset. Additional investigations should be designed to examine how soon after an ischemic episode melatonin must be given to be beneficial. As with any potentially protective agents, in a real-life situation it is usually applied at some interval after a stroke patient is identified. To define what is referred to as the "window of opportunity" during which the protective agent provides benefit is important to its most effective use. This information is not yet available for melatonin.

Another aspect of melatonin’s use is its potential as a prophylactic agent in reducing the vulnerability of the brain (or any other organ) to I/R damage. As noted previously, because of the gradual reduction in endogenous melatonin production throughout life, circulating values of this protective agent are typically diminished in the elderly (90–92). Since the total antioxidant status of the blood of humans correlates with the melatonin concentration in this fluid (Fig. 4; Ref. 123), it is likely that more extensive oxidative damage occurs, such as in vascular tissue, leading to the likelihood of vessel rupture (which causes hemorrhagic stroke) in the brain (and elsewhere).

View larger version (27K): [in this window] [in a new window]   Figure 4. Age-related changes in day and night melatonin levels and the total antioxidant status (TAS) of the blood of humans at various ages. In this study, the individuals, ranging in age from 2 to 89 years, were categorized into 10-year bins. As described previously, increased age is associated with a reduction in nocturnal melatonin values; this drop correlates with a reduction in the TAS of the blood. This suggests that, as melatonin is lost, the ability of individuals to resist oxidative damage is likewise lessened. From Benot et al. (123).

View larger version (16K): [in this window] [in a new window]   Figure 5. Some of the actions of melatonin that contribute to its ability to limit tissue destruction that is a consequence of ischemia/reperfusion injury in the brain as well as in other tissues. Besides scavenging both oxygen- and nitrogen-based reactants, melatonin stimulates (or preserves) the activities of a variety of antioxidative enzymes, such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. Furthermore, it has beneficial actions at the mitochondrial level, where it seemingly enhances the efficiency of the electron transport chain. Also, it limits adhesion molecule formation, thereby reducing the likelihood of clot formation.

	Aspects of Melatonin’s Antioxidative Actions

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

Melatonin is an endogenously generated molecule that is, therefore, not foreign to the body. Under basal conditions, melatonin’s primary site of metabolism is probably the liver, where it is enzymatically converted to 6-hydroxymelatonin, which is subsequently conjugated to sulfuric or glucoronic acid, particularly the former. After their release into the blood as 6-hydroxymelatonin sulfate and 6-hydroxymelatonin glucoronate, respectively, these metabolites are excreted into the urine (34). In addition to these enzymatically mediated metabolic pathways, there may well be alternate routes for the breakdown of melatonin that do not require enzymatic interventions and that, under especially high oxidative stress conditions, produce metabolites that have important functions in the organism. Some of these products include cyclic-3-hydroxymelatonin, AMK, and AFMK (79–82, 125). Importantly, these byproducts, particularly AMK and AFMK, as well as 6-hydroxymelatonin (and possibly others that remain unidentified), are themselves significant reducing agents that limit the degree of oxidative damage under a number of circumstances (83–86, 126, 127).

The ability of melatonin as well as some of its metabolites to function in the capacity of free radical scavengers and/or antioxidants has been referred to as an antioxidant cascade (19). Thus, as mentioned previously, not only is the parent molecule melatonin a protective agent against oxidative damage but, likewise, so are its metabolic "offspring." This combination of cascade actions would seem to increase melatonin’s efficiency in limiting free radical mutilation.

An argument that could justifiably be levied against melatonin as an important antioxidant in vivo is its seemingly low concentrations relative to those of some other free radical scavengers, such as intracellular glutathione. Despite its seemingly low levels, surgical removal of the pineal gland, as noted in this report, a major source of circulating melatonin but by no means the only site of its generation, has repeatedly been shown to aggravate the amount of oxidatively damaged molecules under both basal and elevated oxidative stress conditions (32, 62, 128). This implies that physiological concentrations of melatonin, in conjunction with its metabolites, are sufficiently high to scavenge at least some of the toxic reactants formed or that there are means beyond their scavenging actions that account for some of the protection afforded by the indoleamine. As previously mentioned, subcellular concentrations of melatonin generally correlate poorly with levels in the blood, and certainly a number of fluids have melatonin concentrations that greatly exceed those in the serum (38, 39, 49–53).

It is an interesting possibility that melatonin may actually be inducible under high oxidative stress conditions in mammalian cells. This has been shown to be the case for the unicell Gonyaulax polyedra, where the induction of elevated free radical generation (because of their exposure to 0.08 mM H₂O₂) was associated with a marked rise in intracellular concentrations of melatonin (53). The exceptionally high intracellular levels of the indoleamine in this case were very likely generated for the purpose of inactivating the inordinately large number of ROS being produced as a consequence of the exposure of the cells to H₂O₂.

The question as to whether some mammalian cells have retained the capacity to respond to high oxidative stress conditions with an augmented production of the antioxidant melatonin has never been examined. Of interest regarding this point are the observations of Stefulj and colleagues (44). This group showed, using the reverse transcription polymerase chain reaction method, that the mRNAs for the two enzymes that convert serotonin to melatonin—serotonin-N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT)—are present at low levels in a variety of mammalian cells (e.g., in raphe nuclei of the brain stem, spinal cord, and striatum as well as a variety of nonneuronal tissues). One obvious implication of these findings is that, in fact, even under basal conditions, a variety of mammalian cells may be capable of producing melatonin exclusively for their own use and/or that of their neighbors. No one, however, has yet documented whether melatonin is actually produced in these diverse mammalian cells and, if it is, whether levels exhibit a compensatory rise in response to elevated oxidative stress conditions. These are key presumptions that are worthy of investigation.

That melatonin is rapidly used, presumably as a free radical scavenger, when animals are subjected to raised oxidative stress conditions is known. Thus, when rats are forced to swim at night in darkness when the pineal gland is producing and secreting copious quantities of melatonin, the highly elevated plasma values of the indoleamine drop precipitously, as if the animals were acutely exposed to light. Simultaneously, pineal melatonin concentrations drop, but NAT activity, which rate-limits melatonin production, remains high, as it normally is during darkness (129–133). The precipitous drop in melatonin levels in both the blood and the pineal, despite its high level of production within the gland, is consistent with its rapid uptake and utilization by peripheral tissues. A likely possibility is that the augmented production of free radicals resulting from the physical and psychological stress imposed by swimming causes the sudden utilization of the indoleamine as an antioxidant with the result that blood and pineal levels drop even though its synthesis remains elevated.

	Concluding Remarks

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

 
Numerous studies have now documented that melatonin is a highly effective agent to reduce tissue loss and neurophysiological deficits associated with I/R in animal models. The ability of melatonin to limit cellular destruction as a consequence of the transitory interruption of the blood supply to an organ is not confined to the brain but occurs in other tissues as well, such as the heart (26). Since both stroke and heart attack are very serious conditions that have marked individual and societal consequences, the use of melatonin to potentially protect against these conditions should be seriously considered. The fact that melatonin is an endogenously produced molecule should enhance its safety, given that it is not a foreign substance in the body. Many studies have examined pharmacological doses of melatonin, acutely and chronically in both mammals and humans, as to its toxicity. These investigations have uniformly revealed that the side effects of melatonin are minimal (134–139).

Melatonin’s clinical use for the purpose described herein (or for any use) is hampered by the fact that it is a naturally occurring, nonpatentable molecule. Because of this and because it is easily synthetically produced, it is inexpensive. Thus, the profit margin, even for its widespread use, is limited. This prevents companies and/or individuals from promoting or using this molecule. Considering melatonin’s high efficacy in reducing oxidative damage in animals and its marked potential for improving human health, it should be earnestly considered for use in clinical trials, either as a single molecule and/or in combination with other antioxidants (140).

	References

Top Abstract Introduction Melatonin: Relation to the... Use of Antioxidants to... Aspects of Melatonin’s... Concluding Remarks References

 

1. Kawachi I, Colditz GA, Stampfer MJ, Willett WC, Mason JE, Rosner B, Speizer FE, Hennekens CH. Smoking cessation and decreased risk of stroke in women. J Am Med Assoc 269:232–236, 1993.[Abstract/Free Full Text]
2. Mac Mahon S, Rodgers A. The epidemiological association between blood pressure and stroke: implications for primary and secondary prevention. Hyperten Res 17 (Suppl):523–532, 1994.
3. Wolf PA. Epidemiology and risk factor management. In: Welch KMA, Caplan LR, Reis DJ, Siesjö BK, Weir B, Eds. Primer on Cerebrovascular Diseases. San Diego: Academic Press, pp751–757, 1997.
4. Ginsburg MD. Animal models of global and focal cerebral ischemia. In: Welch KMA, Caplan LR, Reis DJ, Siesjö BK, Weir B, Eds. Primer on Cerebrovascular Diseases. San Diego: Academic Press, pp124–126, 1997.
5. Mazziotta J, Cohen M, Toga A. The measurement of cerebral blood flow and metabolism in human subjects. In: Welch KMA, Caplan LR, Reis DJ, Siesjö BK, Weir B, Eds. Primer on Cerebrovascular Diseases. San Diego: Academic Press, pp34–37, 1997.
6. Pulsinelli WA. Selective neuronal vulnerability: morphological and molecular characteristics. Prog Brain Res 53:29–37, 1985.
7. Pulsinelli WA. Selective neuronal vulnerability and infarction in cerebrovascular disease. In: Welch KMA, Caplan LR, Reis DJ, Siesjö BK, Weir B, Eds. Primer on Cerebrovascular Diseases. San Diego: Academic Press, pp104–107, 1997.
8. Nagel E, Meyer zu Vilsendorf A, Bontels M, Pichlmayr R. Antioxidant vitamins in prevention of ischemia/reperfusion injury. Int J Vit Nutr Res 67:298–306, 1997.
9. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine (3rd ed.). Oxford: Oxford University Press, 2000.
10. Hassman KA. Mechanisms of ischemic injury: is glutamate involved? In: Krieglstein J, Oberpichler-Schwenk H, Eds. Pharmacology of Cerebral Ischemia. Stuttgart: Medpharm Publishers, pp239–251, 1994.
11. Globus MYT, Prado R, Sanchez-Ramos J, Dietrich WD, Ginsberg MD, Busto R. Excitotoxic and ischemic damage: role of nitric oxide. In: Krieglstein J, Oberpichler-Schwenk H, Eds. Pharmacology of Cerebral Ischemia. Stuttgart: Medpharm Publishers, pp363–374, 1994.
12. Hayashi N, Otuki R, Tamura A. Effects of MS-153 on free radical reactions in cerebral ischemia. In: Krieglstein J, Oberpichler-Schwenk H, Eds. Pharmacology of Cerebral Ischemia. Stuttgart: Medpharm Publishers, pp361–365, 1994.
13. Choi DW. Calcium-mediated neurotoxicity: relationship to specific channel types and role of ischemic damage. Trends Neurosci 11:465–469, 1988.[Medline]
14. Katayama Y, Kawamata T, Maeda T, Ishikawa K, Tsubokawa T. Inhibition of the early phase of free fatty acid liberation during cerebral ischemia by excitatory amino acid antagonist administered by microdialysis. Brain Res 635:331–334, 1994.[Medline]
15. Hardeland R, Reiter RJ, Poeggeler B, Tan DX. The significance of the metabolism of the neurohormone melatonin: antioxidative protection and formation of bioactive substances. Neurosci Biobehav Res 17:347–357, 1993.[Medline]
16. Poeggeler B, Reiter RJ, Tan DX, Chen LD, Manchester LC. Melatonin, hydroxyl radical-mediated oxidative damage and aging: a hypothesis. J Pineal Res 14:151–158, 1993.[Medline]
17. Reiter RJ. Functional aspects of the pineal hormone melatonin in combatting cell and tissue damage induced by free radicals: a review. Eur J Endocrinol 134:412–420, 1996.[Abstract/Free Full Text]
18. Reiter RJ, Tan DX, Manchester LC, Qi W. Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence. Cell Biochem Biophys 34:237–256, 2001.[Medline]
19. Tan DX, Reiter RJ, Manchester LC, Yan MT, El-Sawi M, Sainz RM, Mayo JC, Kohen R, Allegra M, Hardeland R. Chemical and physical properties and potential mechanism: melatonin as a broad-spectrum antioxidant and free radical scavenger. Curr Top Med Chem 2:181–198, 2002.[Medline]
20. Allegra M, Reiter RJ, Tan DX, Gentile C, Tesoriere L, Livrea MA. The chemistry of melatonin’s interaction with reactive species. J Pineal Res 34:1–10, 2003.[Medline]
21. Reiter RJ, Tan DX, Osuna C, Gitto E. Actions of melatonin in the reduction of oxidative stress: a review. J Biomed Sci 7:444–458, 2001.
22. Acua-Castroviejo D, Martin M, Macias M, Escames G, Leon J, Khaldy H, Reiter RJ. Melatonin, mitochondria, and cellular bioenergetics. J Pineal Res 30:65–74, 2001.[Medline]
23. Acua-Castroviejo D, Escames G, Carozo A, Leon J, Khaldy H, Reiter RJ. Melatonin, mitochondrial homeostasis and mitochondrial-related diseases. Curr Top Med Chem 2:133–152, 2002.[Medline]
24. Rodriquez C, Mayo JC, Sainz RM, Antolin I, Herrera F, Martin V, Reiter RJ. Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res 36:1–9, 2004.[Medline]
25. Reiter RJ. Melatonin: lowering the high price of free radicals. News Physiol Sci 15:246–250, 2001.
26. Reiter RJ, Tan DX. Melatonin: a novel protective agent against oxidative injury of the ischemic/reperfused heart. Cardiovasc Res 58:10–19, 2003.[Abstract/Free Full Text]
27. Sewerynek E, Reiter RJ, Melchiorri D, Ortiz GG, Lewinski A. Oxidative damage in the liver induced by ischemia-reperfusion: protection by melatonin. Hepatogastroenterology 43:898–905, 1996.[Medline]
28. Okatani Y, Wakatsuki A, Reiter RJ, Enzan H, Miyahara Y. Protective effect of melatonin against mitochondrial injury induced by ischemia and reperfusion of rat liver. Eur J Pharmacol 469:145–152, 2003.[Medline]
29. Brzozowski T, Konturek PC, Konturek SJ, Pajdo R, Bielanski W, Brzozowski, Stachura J, Hahn EG. The role of melatonin and L-tryptophan in prevention of acute gastric lesions induced by stress, ethanol, ischemia and aspirin. J Pineal Res 23:79–89, 1997.[Medline]
30. Inci I, Inci D, Dutly A, Boehler A, Weder W. Melatonin attenuates post transplant lung ischemia-reperfusion injury. Ann Thorac Surg 73:220–225, 2002.[Abstract/Free Full Text]
31. Kilic E, Özdemir YG, Bolay H, Kelestimur H, Dalkara I. Pinealectomy aggravates and melatonin administration attenuates brain damage in focal ischemia. J Cerebr Blood Flow Metab 19:511–516, 1999.[Medline]
32. Kilic E, Hermann DM, Isenmann S, Böhr M. Effects of pinealectomy and melatonin on the retrograde degeneration of retinal ganglion cells in a novel model of intraorbital optic nerve transection in mice. J Pineal Res 32:106–111, 2002.[Medline]
33. Sener G, Sehirli AO, Keyer-Uysal M, Arbak S, Ersoy Y, Yegen BC. The protective effect of melatonin on renal ischemia-reperfusion injury in the rat. J Pineal Res 32:120–126, 2002.[Medline]
34. Reiter RJ. Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12:151–180, 1991.[Abstract/Free Full Text]
35. Faillace MP, Cutrera R, Keller-Sarmento MI, Rosenstein RE. Evidence for local synthesis of melatonin in golden hamster retina. Neuroreport 6:2093–2095, 1995.[Medline]
36. Huether G, Poeggeler B, Reimer A, George A. Effect of tryptophan administration on circulating melatonin levels in checks and rats: evidence for stimulation of melatonin synthesis and release in the gastrointestinal tract. Life Sci 52:945–953, 1992.
37. Bubenik GA. Gastrointestinal melatonin: localization, function and clinical relevance. Dig Dis Sci 47:2336–2348, 2002.[Medline]
38. Tan DX, Manchester LC, Reiter RJ, Qi W, Zhang M, Weintraub ST, Cabrera J, Sainz RM, Mayo JC. Identification of highly elevated levels of melatonin in bone marrow: its origin and significance. Biochim Biophys Acta 1472:206–214, 1999.[Medline]
39. Conti A, Conconi S, Hertens E, Skwarlo-Sonta K, Markowska M, Maestroni GJM. Evidence for melatonin synthesis in mouse and human bone marrow cells. J Pineal Res 28:193–202, 2000.[Medline]
40. Carrillo-Vico A, Calvo JR, Abreu P, Lardone PJ, Garcia-Morino S, Reiter RJ, Guerrero JM. Evidence of melatonin synthesis in human lymphocytes and its physiological significance: possible role as intracrine, autocrine and/or paracrine substance. FASEB J 18:537–539, 2004.[Abstract/Free Full Text]
41. Slominski A, Pisarchik A, Semak I, Sweatman T, Wortsman J, Szczesniewski A, Slugocki G, McNulty J, Kauser S, Tobin DI, Jing C, Johansson O. Serotoninergic and melatoninergic systems are fully expressed in human skin. FASEB J 16:896–898, 2002.[Abstract/Free Full Text]
42. Lopez-Gonzalez MA, Guerrero JM, Delgado F. Presence of the pineal hormone in rat cochlea: its variations with lighting conditions. Neurosci Lett 238:81–83, 1997.[Medline]
43. Abe M, Itoh MT, Miyata M, Shimizu K, Sumi Y. Circadian rhythm of serotonin N-acetyltransferase activity in rat lens. Exp Eye Res 70:805–808, 2000.[Medline]
44. Stefulj J, Hörtner M, Ghosh M, Schauenstein K, Rinner I, Wölfer A, Semmler J, Liebmann PM. Gene expression of the key enzymes of melatonin synthesis in extrapineal tissues of the rat. J Pineal Res 30:243–247, 2001.[Medline]
45. Hattori A, Migitaka H, Iigo M, Itoh M, Yamamoto K, Oktani-Kandro R, Hara M, Suzuki T, Reiter RJ. Identification of melatonin in plants: its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem Mol Biol Int 35:627–634, 1995.[Medline]
46. Reiter RJ, Tan DX, Burkhardt S, Manchester LC. Melatonin in plants. Nutr Rev 59:286–290, 2001.[Medline]
47. Tan DX, Manchester LC, Hardeland R, Lopez-Burillo S, Mayo JC, Sainz RM, Reiter RJ. Melatonin: a hormone, a tissue factor, an autocoid, a paracoid, and an antioxidant vitamin. J Pineal Res 34:75–78, 2003.[Medline]
48. Reiter RJ, Vaughan MK, Blask DE. Possible role of the cerebrospinal fluid in the transport of pineal hormones in mammals. In: Knigge KM, Scott DE, Koboyashi H, Ishii S, Eds. Brain-Endocrine Interaction II. Basel: Karger, pp337–354, 1975.
49. Skinner DC, Malpaux B. High melatonin concentrations in third ventricular cerebrospinal fluid are not due to Galen vein blood recirculating through the choroid plexus. Endocrinology 140:4399–4405, 1999.[Abstract/Free Full Text]
50. Tricoire H, Locatelli A. Chemineau P, Malpaux B. Melatonin enters cerebrospinal fluid through the pineal recess. Endocrinology 143:184–190, 2002.
51. Tan DX, Manchester LC, Reiter RJ, Qi W, Hanes MA, Farley NJ. High physiological levels of melatonin in bile of mammals. Life Sci 65:2523–2529, 1999.[Medline]
52. Brzezinski A, Seibel MM, Lynch HJ, Deng MH, Wurtman RJ. Melatonin in human preovulatory follicular fluid. J Clin Endocrinol Metab 64:865–867, 1987.[Abstract/Free Full Text]
53. Antolin I, Obst B, Burkhardt S, Hardeland R. Antioxidant properties in a high-melatonin organism: the dinoflagellate Gonyaulax polyedra is rescued from lethal oxidative stress by strongly elevated, but physiologically possible concentrations of melatonin. J Pineal Res 23:182–190, 1997.[Medline]
54. Spencer J, Hardeland R, Fuhrberg B. Melatonin and other methoxylated indoles in yeast: presence in high concentrations and dependence on tryptophan availability. Cytologia 64:209–213, 1999.
55. Martin M, Macias M, Escames G, Leon J, Acua-Castroviejo D. Melatonin but not vitamins C and E maintain glutathione hemostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress. FASEB J 14:1677–1679, 2000.[Abstract/Free Full Text]
56. Menendez-Pelaez A, Poeggeler B, Reiter RJ, Barlow-Walden L, Pablos MI, Tan DX. Nuclear localization of melatonin in different mammalian tissues: immunocytochemical and radioimmunoassay evidence: J Cell Biochem 53:373–382, 1993.[Medline]
57. Reiter RJ, Tan DX. What constitutes a physiological concentration of melatonin? J Pineal Res 34:79–80, 2003.[Medline]
58. Menendez-Pelaez A, Reiter RJ. Distribution of melatonin in mammalian tissues: relative importance of nuclear verses cytosolic localization. J Pineal Res 15:59–69, 1993.[Medline]
59. Reiter RJ, Burkhardt S, Cabrera J, Garcia JJ. Beneficial neurobiological effects of melatonin under conditions of increased oxidative stress. Curr Med Chem 2:45–58, 2002.
60. Baydas G, Kutlur S, Naziroglu M, Canpolat S, Sandal S, Ozcan M, Kelestimur H. Inhibitory effect of melatonin on neural lipid peroxidation induced by intracerebroventricularly administered homocysteine. J Pineal Res 34:36–39, 2003.[Medline]
61. Ohta Y, Kongo M, Kishikawa T. Preventive effect of melatonin on the progression of -naphthylisothiocyanate-induced acute liver injury in rats. J Pineal Res 34:185–193, 2003.[Medline]
62. Reiter RJ, Tan DX, Kim SJ, Manchester LC, Qi W, Garcia JJ, Cabrera JC, El-Sokkary G, Rouvier-Garay V. Augmentation of indices of oxidative damage in life-long melatonin-deficient rats. Mech Aging Dev 110:157–173, 1999.
63. Okatani Y, Wakatsuki A, Reiter RJ, Miyohara Y. Melatonin reduces oxidative damage of neural lipids and proteins in senescence-associated mouse. Neurobiol Aging 23:639–644, 2002.[Medline]
64. Sener G, Sehirli AO, Ayanoglu-Dülger G. Protective effect of melatonin, vitamin E and N-acetylcysteine against acetaminophen toxicity in mice: a comparative study. J Pineal Res 35:61–68, 2003.[Medline]
65. Karbownik M, Reiter RJ, Burkhardt S, Tan DX, Lewinski A. Melatonin attenuates estradiol-induced oxidative damage to DNA: relevance to cancer. Exp Biol Med 226:707–712, 2001.[Abstract/Free Full Text]
66. Hsu CH, Chi BC, Casida JE. Melatonin reduces phosphine-induced lipid and DNA oxidation in vitro and in vivo in rat brain. J Pineal Res 32:53–62, 2002.[Medline]
67. Sun FY, Lin X, Mao LZ, Ge W, Zhang LM, Huang YL, 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 33:48–56, 2002.[Medline]
68. Mansouri A, Demeilliers C, Amsellam S, Pessayre D. Fromenty B. Acute ethanol administration damages and depletes mitochondrial DNA in mouse liver, brain, heart and skeletal muscle: protective effects of antioxidants. J Pharmacol Exp Ther 298:737–743, 2001.[Abstract/Free Full Text]
69. Yamamoto HA, Mohanan PV. Ganglioside GT1B and melatonin inhibit brain mitochondrial DNA damage and seizures induced by kainic acid in mice. Brain Res 964:100–106, 2003.[Medline]
70. Poeggeler B, Thuermann S, Rose A. Schoenke M, Burkhardt S, Hardeland R. Melatonin’s unique radical scavenging properties—roles of its functional substituents as revealed by a comparison with its structural analogs. J Pineal Res 33:20–30, 2002.[Medline]
71. Abel Wahab MH, Akoul EEMS, Abdel-Aziz AAH. Modulatory effects of melatonin and vitamin E on doxorubicin-induced cardiotoxicity in Ehrlich acites carcinoma-bearing mice. Tumori 86:157–162, 2000.[Medline]
72. Montilla P, Cruz A, Padillo FJ, Tunez I, Gascon F, Munoz MC, Ganez M, Pera C. Melatonin versus vitamin E as protective treatment against oxidative stress after extra-hepatic bile duct ligation in rats. J Pineal Res 31:138–144, 2001.[Medline]
73. Baydas G, Canataan H, Turkoglu A. Comparative analysis of the protective effects of melatonin and vitamin E on streptozocin-induced diabetes mellitus. J Pineal Res 32:225–230, 2002.[Medline]
74. Tan DX, Chen LD, Poeggeler B, Manchester LC, Reiter RJ. Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr J 1:57–60, 1993.
75. Reiter RJ. Oxidative damage in the central nervous system: protection by melatonin. Prog Neurobiol 56:359–384, 1998.[Medline]
76. Cuzzocrea S, Reiter RJ. Pharmacological action of melatonin in shock, inflammation and ischemia/reperfusion injury. Eur J Pharmacol 426:1–10, 2001.[Medline]
77. Cheung RTF. The utility of melatonin in reducing cerebral damage resulting from ischemia and reperfusion. J Pineal Res 34:153–160, 2003.[Medline]
78. Reiter RJ, Sainz RM, Lopez-Burillo S, Mayo JC, Manchester LC, Tan DX. Melatonin ameliorates neurological damage and neurophysiological deficits in experimental models of stroke. Ann N Y Acad Sci 993:35–47, 2003.[Medline]
79. Hirata F, Hayaishi O, Tokuyama T, Senoh S. In vitro and in vivo formation of two new metabolites of melatonin. J Biol Chem 249:1311–1313, 1974.[Abstract/Free Full Text]
80. Carampin P, Rosan S, Dalzoppo D, Zagotto G, Zatta P. Some biochemical properties of melatonin and the characterization of melatonin of a relevant metabolite arising from its interaction with H₂O₂. J Pineal Res 34:134–142, 2003.[Medline]
81. de Almeida EA, Martinez GR, Klitzke CF, de Medeiros MHG, Mascio PD. Oxidation of melatonin by singlet molecular oxygen (O₂ (1g)) produces N¹-acetyl-N²-formyl-5-methoxykynuramine. J Pineal Res 35:131–137, 2003.[Medline]
82. Rozov SV, Filatova EV, Orlov AA, Valkova AV, Zhloba ARA, Blaskko EL, Pozdeyev NV. N¹-acetyl-N²-formyl-5-methoxykynur-amine is a product of melatonin oxidation in rats. J Pineal Res 35:245–250, 2003.[Medline]
83. Burkhardt S, Reiter RJ, Tan DX, Hardeland R, Cabrera J, Karbownik M. DNA oxidatively damaged by chromium (III) and H₂O₂ is protected by melatonin, N¹-acetyl-N²-formyl-5-methoxykynuramine, resveratrol and uric acid. Int J Biochem Cell Biol 33:775–783, 2001.[Medline]
84. Tan DX, Manchester LC, Burkhardt S, Sainz RM, Mayo JC, Kohen R, Shohami E, Huo YX, Hardeland R, Reiter RJ. N¹-acetyl-N²-formyl-5-methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent antioxidant. FASEB J 15:2294–2296, 2001.[Abstract/Free Full Text]
85. Lopez-Burillo S, Tan DX, Rodriquez-Gallego V, Manchester LC, Mayo JC, Sainz RM. Reiter RJ. Melatonin and its derivatives cyclic 3-hydroxymelatonin, N¹-acetyl-N²-formyl-5-methoxykynuramine and 6-methoxymelatonin reduce oxidative DNA damage induced by Fenton reagents. J Pineal Res 34:178–184, 2003.
86. Ressmeyer AR, Mayo JC, Zelasko V, Sainz RM, Tan DX, Poeggeler B, Antolin I, Zsizsik BK, Reiter RJ, Hardeland R. Antioxidant properties of the melatonin N¹-acetyl-5-methoxykynuramine (AMK): scavenging of free radicals and prevention of protein destruction. Redox Report 8:205–213, 2003.[Medline]
87. Ianas O, Olinescu R, Badescu I. Melatonin involvement in oxidative processes. Endocrinologie 29:147–153, 1991.[Medline]
88. Manev H, Uz T, Kharlamov A, Joo JY. Increased brain damage after stroke or excitotoxic seizures in melatonin-deficient rats. FASEB J 10:1546–1551, 1996.[Abstract]
89. Joo JY, Uz T, Manev H. Opposite effects of pinealectomy and melatonin administration on brain damage following cerebral focal ischemia in rat. Rest Neurol Neurosci 13:185–191, 1998.[Medline]
90. Reiter RJ. The aging pineal and its physiological consequences. Bio Essays 14:169–175, 1992.[Medline]
91. Zhou JN, Liu RY, Heerikhuize JV, Hofman MA, Swaab DF. Alterations in the circadian rhythm of salivary melatonin begin in middle-age. J Pineal Res 34:11–16, 2003.[Medline]
92. Zhou JN, Liu RY, Kamphorst W, Hofman MA, Swaab DF. Early neuropathological Alzheimer’s changes in aged individuals are accompanied by decreased cerebrospinal fluid melatonin levels. J Pineal Res 35:125–130, 2003.[Medline]
93. Manev H, Uz T. The role of the light-dark cycle and melatonin in stroke outcome. J Stroke Cerebrovasc Dis 7:165–167, 1996.
94. Cho S, Joh TH, Baik HH, Dibinis C, Volpe BT. Melatonin administration protects CA1 hippocampal neurons after ischemia in rats. Brain Res 755:335–338, 1997.[Medline]
95. Giusti P, Lipartiti M, Franceschini D, Schiavo N, Floreani M, Manev H. Neuroprotection by melatonin from kainate-induced excitotoxicity in rats. FASEB J 10:891–896, 1996.[Abstract]
96. Lezoualc’h F, Skutella T, Wideman M, Behl C. Melatonin prevents oxidative stress-induced cell death in hippocampal cells. Neuroreport 7:2071–2076, 1996.[Medline]
97. Lezoualc’h F, Sparapani M, Behl C. N-acetylserotonin and melatonin protect neurons against oxidative challenges and suppress the activity of the transcription factor NF-B. J Pineal Res 24:167–178, 1998.
98. Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623–634, 1987.
99. Choi DW. The excitotoxic concept. In: Welch KMA, Caplan LR, Reis DJ, Siesjö BK, Weir B, Eds. Primer on Cerebrovascular Disease. San Diego: Academic Press, pp188–190, 1997.
100. Reiter RJ, Cabrera J, Sainz RM, Mayo JC, Manchester LC, Tan DX. Melatonin as a pharmacological agent against neuron loss in experimental models of Huntington’s disease, Alzheimer’s disease and Parkinsonism. Ann N Y Acad Sci 890:471–485, 2000.
101. Li XJ, Zhang LM, Gu J, Zhang AZ, Sun FJ. Melatonin decreases production of the hydroxyl radical during cerebral ischemia-reperfusion. Acta Pharmacol Sinica 18:394–396, 1997.
102. Leon J, Acua-Castroviejo D, Sainz RM, Mayo JC, Tan DX, Reiter RJ. Melatonin and mitochondrial function. Life Sci(in press).
103. Ling X, Zhang LM, Lu SC, Li XJ, Sun FJ. Protective effect of melatonin on injured cerebral neurons is associated with bcl-2 overexpression. Acta Pharmacol Sin 20:409–414, 1999.
104. Andrabi SA, Sayeed I, Siemen D, Wolf G, Horn TFW. Direct inhibition of the mitochondrial permeability transition pore: a possible mechanism responsible for anti-apoptotic effects of melatonin. FASEB J 18:869–871, 2004.[Abstract/Free Full Text]
105. Jou MJ, Peng TI, Reiter RJ, Jou SB, Wu HY, Wen ST. Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes. J Pineal Res(in press).
106. Sainz RM, Mayo JC, Rodriquez C, Tan DX, Lopez-Burillo S, Reiter RJ. Melatonin and cell death: differential actions on apoptosis in normal and cancer cells. Cell Mol Life Sci 60:1407–1426, 2003.[Medline]
107. Sinha K, Degaonkar MN, Jagannathan NR, Gupta Y. Effect of melatonin on ischemia/reperfusion injury induced by middle cerebral artery occlusion in rats. Eur J Pharmacol 428:185–192, 2002.
108. Kondoh T, Uneyama H, Nishino H, Torii K. Melatonin reduces cerebral edema formation caused by transient forebrain ischemia in rats. Life Sci 72:583–590, 2002.[Medline]
109. Torii K, Uneyama H, Nishino H, Kondoh T. Melatonin suppresses cerebral edema caused by middle cerebral artery occlusion/reperfusion in rats assessed by magnetic resonance imaging. J Pineal Res 36:18–24, 2004.[Medline]
110. Borlongan CV, Yamamoto M, Takei N, Kumazahi M, Ungsuparkorn C, Hida H, Sanberg PR, Nishino H. Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia. FASEB J 14:1307–1317, 2000.[Abstract/Free Full Text]
111. Baydas G, Reiter RJ, Nedzvetskii VS, Nurush PA, Kirichendo SV. Altered glial fibrillary acidic protein content and its degradation in the hippocampus, cortex and cerebellum of rats exposed to constant light: reversal by melatonin. J Pineal Res 33:134–139, 2002.[Medline]
112. Baydas G, Reiter RJ, Yasar A, Tuzcu M, Gokdemir I, Nedzvetskii VS. Melatonin reduces glial cell reactivity in the hippocampus, cortex and cerebellum of streptozotocin-induced diabetic rat. Free Radic Biol Med 35:797–804, 2003.[Medline]
113. Kilic E, Kilic V, Yulug B, Hermann DM, Reiter RJ. Melatonin reduces disseminate neuronal death after mild focal ischemia in mice via inhibition of caspase-3 and is suitable as an add-on treatment to tissue-plasminogen activator. J Pineal Res 36:171–176, 2004.[Medline]
114. Cuzzocrea S, Costantino G, Gitto E, Mazzon E, Fulia F, Serraino I, Cordaro S, Barberi I, De Sarro A, Caputi AP. Protective effects of melatonin in ischemic brain injury. J Pineal Res 29:217–227, 2000.[Medline]
115. Guerrero JM, Reiter RJ, Ortiz GG, Pablos MI, Sewerynek E, Chuang JI. Melatonin prevents increases in neural nitric oxide and cyclic GMP production after transient brain ischemia and reperfusion in the Mongolian gerbil (Meriones unguiculatus). J Pineal Res 23:24–31, 1997.[Medline]
116. Pei Z, Ho HT, Cheung RT. Pretreatment with melatonin reduces volume of cerebral infarction in a permanent middle cerebral artery occlusion stroke model in the rat. Neurosci Lett 318:141–144, 2002.[Medline]
117. Pei Z, Pang SF, Cheung RP. Pretreatment with melatonin reduces volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model. J Pineal Res 32:168–172, 2002.[Medline]
118. Pei Z, Pang SF, Cheung RT. Administration of melatonin after onset of ischemia reduces the volume of cerebral infarction in a middle cerebral artery occlusion stroke model. Stroke 34:770–775, 2003.[Abstract/Free Full Text]
119. Pei Z, Fung PC, Cheung RT. Melatonin reduces nitric oxide level during ischemia but not blood-brain-barrier breakdown during reperfusion in a rat middle cerebral artery occlusion stroke model. J Pineal Res 34:110–118, 2003.[Medline]
120. Wakatsuki A, Okatani Y, Izumiya C, Ikenoue N. Melatonin protects against ischemia and reperfusion-induced oxidative and lipid DNA damage in fetal rat brain. J Pineal Res 26:147–152, 1999.[Medline]
121. Okatani Y, Okamoto K, Hayachi K, Wakatsuki A, Sagara Y. Maternal-fetal transfer of melatonin in human pregnancy near term. J Pineal Res 25:129–134, 1998.[Medline]
122. Letechipia-Vallejo G, Gonzalez-Burgos I, Cervantes M. Neuro-protective effect of melatonin on brain damage induced by acute global cerebral ischemia in cats. Arch Med Res 32:186–192, 2001.[Medline]
123. Benot S, Goberna R, Reiter RJ, Garcia-Moreno S, Osuna C, Guerrero JM. Physiological levels of melatonin correlate with the antioxidant capacity of human serum. J Pineal Res 27:59–64, 1999.[Medline]
124. Cuzzocrea S, Costantino G, Mazzon R, Micali A, Sarro A, Caputi AP. Beneficial effects of melatonin in a rat model of splanchnic artery occlusion and reperfusion. J Pineal Res 28:52–63, 2000.[Medline]
125. Tan DX, Manchester LC, Reiter RJ, Plummer BF, Hardies LJ, Weintraub ST, Vijayalaxmi, Shepherd AM. A novel melatonin metabolite, cyclic 3-hydroxymelatonin: a biomarker of in vivo hydroxyl radical generation. Biochem Biophys Res Commun 253:614–620, 1998.[Medline]
126. Hara M, Iigo M, Okatani-Kaneko R, Nakamura N, Suzuki T, Reiter RJ, Hirata K. Administration of melatonin and related indoles prevents exercise-induced cellular oxidative changes in rats. Biol Signals 6:90–100, 1997.[Medline]
127. Maharaj DS, Walker RB, Glass BD, Daya S. 6-hydroxymelatonin protects against cyanide induced oxidative stress in rat brain homogenates. J Chem Neuroanat 26:103–107, 2003.[Medline]
128. Shaper SD, Floreani M, Ceccon M, Facci L, Giusti P. Excitotoxicity, oxidative stress and the neuroprotective potential of melatonin. Ann N Y Acad Sci 890:107–118, 1999.[Medline]
129. Troiani ME, Reiter RJ, Vaughan MK, Oakin S, Vaughan GM. Swimming depresses nighttime melatonin content without changing N-acetyltransferase activity in the rat pineal gland. Neuroendocrinology 47:55–60, 1987.
130. Wu W, Reiter RJ, Troiani ME, Vaughan GM. Elevated daytime rat pineal and serum melatonin levels induced by isoproterenol are depressed by swimming. Life Sci 41:1473–1479, 1987.[Medline]
131. Troiani ME, Reiter RJ, Tannenbaum MG, Puig-Domingo M, Guerrero JM, Menendez-Pelaez A. Neither the pituitary gland nor the sympathetic nervous system is responsible for eliciting the large drop in elevated rat pineal melatonin levels due to swimming. J Neural Transm 74:149–160, 1988.
132. Ueck M, Troiani ME, Reiter RJ. Transient reduction in pineal melatonin levels but not N-acetyltransferase activity in rats forced to swim for 15 minutes at night. Neuroendocr Lett 10:81–90, 1988.
133. Reiter RJ, Richardson BA. Some perturbations that disturb the circadian melatonin rhythm. Chronobiol Int 9:314–321, 1992.[Medline]
134. Nordlund JJ, Lerner AB. The effects of oral melatonin on skin color and on the release of pituitary hormones. J Clin Endocrinol Metab 45:768–774, 1977.[Abstract/Free Full Text]
135. Jahnke G, Marr M, Myers C, Wilson R, Travlos G, Price C. Maternal and developmental toxicity of melatonin administered orally to pregnant Sprague-Dawley rats. Toxicol Sci 50:271–279, 1999.[Abstract/Free Full Text]
136. Jan JE, Hamilton D, Seward N, Fast DK, Freeman RD, Laudon M. Clinical trials of controlled-release melatonin in children with sleep-wake cycle disorders. J Pineal Res 29:34–39, 2000.[Medline]
137. Gitto E, Karbownik M, Reiter RJ, Tan DX, Cuzzocrea S, Chiurazzi S, Cordaro S, Corona G, Trimarchi G, Barberi I. Effects of melatonin treatment in septic newborns. Pediatr Res 50:756–760, 2001.[Medline]
138. Gitto E, Reiter RJ, Amodio A, Romero 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 36:250–255, 2004.[Medline]
139. Gitto E, Romeo C, Reiter RJ, Inpellizzeri R, Pesce S, Basile M, Autonuccio P, Trimarchi G, Gentile C, Barberi I, Zuscaselto B. Melatonin reduces oxidative stress in surgical neonates. J Pediatr Surg 39:184–189, 2004.[Medline]
140. Tan DX, Manchester LC, Sainz RM, Mayo JC, Alvares FL, Reiter RJ. Antioxidant strategies in protection against neurodegenerative disorders. Curr Opin Ther Patents 13:1513–1543, 2003.

This article has been cited by other articles:

				G. Jin, K. Arai, Y. Murata, S. Wang, M. F. Stins, E. H. Lo, and K. van Leyen Protecting Against Cerebrovascular Injury: Contributions of 12/15-Lipoxygenase to Edema Formation After Transient Focal Ischemia Stroke, September 1, 2008; 39(9): 2538 - 2543. [Abstract] [Full Text] [PDF]

				E. B. C. Llambias, M. B. Mazzetti, S. M. Lelli, C. Aldonatti, and L. C. San Martin de Viale Melatonin Formation in Pineal Gland from Rats with Hexachlorobenzene Experimental Porphyria International Journal of Toxicology, November 1, 2007; 26(6): 545 - 551. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Reiter, R. J. Articles by Kilic, E. Search for Related Content PubMed PubMed Citation Articles by Reiter, R. J. Articles by Kilic, E.