| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Institute of Pharmacology and Toxicology, Medical Faculty Charité, Humboldt-University Berlin, D-10098 Berlin, Germany; and
Department of Neurology, Medical Faculty Charité, Humboldt-University Berlin, D-10098 Berlin, Germany
1Correspondence: Institute of Pharmacology and Toxicology, Medical Faculty Charité, Dorotheenstr. 94, D-10098 Berlin, Germany. E-mail: heide.hoertnagl{at}charite.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: ethylcholine aziridinium (AF64A) • staurosporine • oxygen-glucose deprivation • cortex • oxidative stress
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3
). Neuroprotective effects of
melatonin have been demonstrated mainly in models of neuronal cell
death in which oxygen free radicals or excitotoxins are involved. In
the N-methyl-4-phenylpyridinium and 6-hydroxydopamine (6-OHDA) models
of Parkinson’s disease, melatonin completely reversed the rises in
lipid peroxidation products, the decrease in tyrosine hydroxylase in
striatum and substantia nigra, and rescued dopamine neurons in culture
(4
5
6
7)
. Melatonin also prevented kainate-induced neuronal
cell death and reduced lipid peroxidation products in rats and mice
in vivo (8
9
10)
. Furthermore, melatonin
protects against glutamate-induced cell death in the clonal hippocampal
cell line HT22 (11)
, prevents delayed neuronal death
induced by enhanced excitatory transmission in hippocampal pyramidal
neurons in culture (12)
, and rescues neuroblastoma cells
exposed to toxic fragments of Alzheimer’s ß-amyloid
(13)
. An anticonvulsant activity of melatonin has been
demonstrated against excitotoxin-induced seizures by quinolinate,
kainate, and glutamate in mice and by iron or amygdala kindling in rats
(14
15
16
17)
. The occurrence of increased brain damage after
stroke or excitotoxic seizures in melatonin-deficient rats is in line
with these findings (18)
.
Besides the antioxidant potential, several other mechanisms are
considered to be involved in the neuroprotection mediated by melatonin,
including interactions with calmodulin (19
, 20)
and
microtubular components (20)
, blockade of increases in
intracellular Ca2+ levels (13)
,
maintenance of cellular glutathione homeostasis (21)
,
inhibition of activation of NF-
B by cytokines such as tumor necrosis
factor 
(22)
, inhibition of the expression of inducible
nitric oxide synthase at the transcriptional level (23)
,
and changes in gene expression of antioxidant enzymes
(24)
.
The aim of the present study was to answer the question of whether
melatonin is also neuroprotective in the case of neuronal damage, which
is not primarily related to oxidative stress or excitotoxicity. For
this purpose, we investigated the efficacy of melatonin in primary
neuronal cultures by using three different models of apoptotic,
excitotoxin-independent neurodegeneration in comparison with necrotic
neuronal damage. Oxygen-glucose deprivation (OGD) was applied as a
model for excitotoxic and oxidative stress inducing cell damage. A
protective effect of melatonin against hypoxia/reoxygenation has been
reported in primary cultures of rat cortical neurons (25)
and after transient forebrain ischemia in rats (26)
. As a
standard apoptotic model treatment with staurosporine was used. The
mycotoxin staurosporine is believed to activate programmed cell death
in virtually all cells (27
28
29)
. It has also been shown to
induce neuronal apoptosis in primary neuronal cultures (30
, 31)
. In addition, a model characterized by a delayed occurrence
of apoptotic cell death induced by the neurotoxin ethylcholine
aziridinium (AF64A) was applied. AF64A, initially introduced as a model
of cholinergic hypofunction (for a recent review, see ref
32
), has been shown to initiate neuronal cell death
in vivo and in vitro by activating mechanisms of
apoptosis (33)
. An involvement of glutamate in the
neurotoxicity of AF64A has been excluded (34)
. In primary
neuronal cultures of various brain areas, AF64A induces neuronal cell
death with a delay of 2–3 days (35)
. The apoptotic
component of OGD unmasked by glutamate antagonists (36)
served as a third model.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-phenylnitrone (PBN),
dimethylthiourea (DMTU), MTT, and enzyme standard for kinetic lactate
dehydrogenase (LDH) test were obtained from Sigma Chemie GmbH
(Deisenhofen, Germany), neurobasal medium and supplement B27 from Life
Technologies, Inc. (Eggenstein, Germany); modified Eagle‘s medium,
phosphate-buffered saline (PBS), HEPES buffer, trypsin/EDTA,
penicillin/streptomycin, L-glutamine, collagen-G, and poly-L-lysine
from Biochrom (Berlin, Germany); multiwell plates from Falcon (Franklin
Lakes, N.J.); the caspase inhibitor Z-VAD-FMK from
Calbiochem-Novabiochem (Bad Soden/Taunus, Germany). AF64A was prepared
from acetylethylcholine mustard (Research Biochemicals
International, Natick, Mass.) according to Fisher et al.
(37)
.
Primary neuronal cell culture
Primary neuronal cultures of cerebral cortex were obtained from
embryos (E16-E18) of Wistar rats (Bundesinstitut für
gesundheitlichen Verbraucherschutz und Veterinärmedizin, Berlin,
Germany). Cultures were prepared according to Brewer (38)
with the following modifications. Cerebral cortex was dissected,
incubated for 15 min in trypsin/EDTA (0.05/0.02% w/v in PBS) at
36.5°C, rinsed twice with PBS and once with dissociation medium
(modified Eagle‘s medium with 10% fetal calf serum, 10 mM HEPES, 44
mM glucose, 100 U penicillin + streptomycin/ml, 2 mM L-glutamine, 100
IE insulin/l), dissociated by Pasteur pipette in dissociation medium,
pelleted by centrifugation (at 210 g for 2 min at 21°C),
redissociated in starter medium (neurobasal medium with supplement B27,
100 U penicillin + streptomycin/ml, 0.5 mM L-glutamine, 25 µM
glutamate), and plated in 24-well plates or 6-well plates in a density
of 200,000 cells per cm2. Wells were pretreated
by incubation with poly-L-lysine (0.5% w/v in PBS) for 1 h at
room temperature, then rinsed with PBS, followed by incubation with
coating medium (dissociation medium with 0.03
w/v collagen G) for
1 h at 37°C, then rinsed twice with PBS before seeding cells in
starter medium. Cultures were kept at 36.5°C and 5%
CO2 and fed beginning with the fourth day
in vitro (DIV) with cultivating medium (starter medium
without glutamate) by replacing half of the medium twice a week.
Injury paradigms
In all models the serum-free primary neuronal cultures were
treated after 10 to 14 DIV. Condition of cells at various time points
after treatment was determined morphologically by phase contrast
microscopy.
AF64A (5–80 µM) exposure was either for 5 h with subsequent rinsing and reapplication of conditioned medium or AF64A was not removed during the 72 h observation period. Controls received an equivalent amount of vehicle and were rinsed correspondingly.
Staurosporine was dissolved in DMSO (10 mM stock solution) and diluted with PBS to give the final concentrations of 100 and 300 nM in culture. The vehicle-treated cultures received the same amount of DMSO (in PBS) present in the highest dose of staurosporine.
For OGD medium was removed from the cultures and preserved. Cultures
were rinsed twice with PBS, then subjected to OGD for 120 min in a
balanced salt solution at pO2 < 2 mm Hg,
followed by replacement of the preserved medium as described previously
(39)
. For standardization of the neuronal damage, the
sister cultures were treated with an excess of glutamate (5 mM; full
kill). To differentiate between the excitotoxic and apoptotic
components of OGD as described previously (36)
, cells were
treated with the NMDA antagonist MK-801 (10 µM) 30 min prior to OGD
(120 or 180 min).
Caspase inhibition
For caspase inhibition the general caspase inhibitor Z-VAD-FMK
(100 µM) dissolved in DMSO (0.2% final concentration) was used. In
the AF64A model Z-VAD-FMK or vehicle containing the corresponding
amount of DMSO was added 1 h before as well as 24 and 48 h
after AF64A application. In the model of OGD plus MK-801 pretreatment
Z-VAD-FMK was added at the beginning of reoxygenation and 24 h
later.
Treatment with melatonin, PBN, and DMTU
Melatonin was dissolved in a 40% ethanol solution, then
diluted with medium to a 20 mM stock solution. It was applied to the
cell cultures in a final concentration of 0.01 to 2.0 mM. Melatonin was
added 1 or 2 h before AF64A, 1 h before staurosporine and
1 h before OGD. The final concentration of ethanol per well did
not exceed 0.01% at all melatonin concentrations used. The
vehicle-treated control cultures received the equal amount of ethanol
(0.01%). PBN (dissolved in DMSO) and DMTU (dissolved in PBS) were
added 1 h before AF64A or staurosporine. The final concentration
of DMSO per well did not exceed 0.2%. Vehicle-treated cultures
received the equal amount of DMSO.
Cell viability assays
Neuronal injury was quantitatively assessed by the measurement
of LDH in the medium (40)
at 24, 48, and 72 h after
AF64A or staurosporine application and at 24 and 48 h after OGD.
As a second measure the MTT assay (41)
, based on the
cleavage of the yellow tetrazolium salt MTT to purple formazan by
mitochondrial enzymes in metabolically active cells was performed
72 h after addition of AF64A or staurosporine.
Measurement of oxidative stress
The extent of oxidative stress was assessed by the amount of
malondialdehyde formed in the cell cultures exposed to AF64A or
staurosporine. For this purpose, medium was removed and cells were
washed with PBS and harvested by scraping off mechanically at various
time points after the addition of staurosporine, AF64A, melatonin, or
the corresponding vehicle. In addition, the effect of the antioxidants
melatonin, PBN, and DMTU on the formation of malondialdehyde induced by
AF64A or staurosporine was investigated. The concentration of
malondialdehyde was measured according to the method of Wong et al.
(42)
.
Data analyses
Data are shown as means ± SE. To avoid
possible variations of the cell cultures depending on the quality of
dissection and seeding procedures, data were pooled from two to three
dissections. For statistical analyses the one-way ANOVA was followed by
Tukey post hoc tests.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, representative phase contrast photomicrographs of primary neuronal
cultures under control conditions after AF64A exposure and after OGD
are summarized. AF64A triggered morphological signs of apoptosis
including degeneration of neurites, shrinkage of cell bodies, and
fragmentation in condensed particles, whereas after OGD (120 min) the
occurrence of blown up cell bodies typical for necrosis prevailed.
|
Effect of melatonin treatment on cell viability in native primary
neuronal cultures
A single dose treatment of primary neuronal cultures with
melatonin (dose range of 0.01 to 0.5 mM) induced a significant increase
in LDH release after 72 h as compared to vehicle-treated cultures
(Table 1
). This effect was not obvious after 24 h, started after 48 h,
and reached significance 72 h after a single exposure to melatonin
(0.05 and 0.5 mM). The increase in LDH release was not due to the
addition of small amounts of ethanol, since vehicle-treated cells
received the equivalent amount of ethanol. Moreover, a dose-dependent
increase in the concentration of malondialdehyde was observed 24 h
after the addition of melatonin (0.5 to 2.0 mM) to native cortical
cells, indicating the occurrence of lipid peroxidation (Fig. 2
).
|
|
Effect of melatonin on staurosporine-induced apoptotic neuronal
cell death
When applied to cortical neuronal cultures, staurosporine
increased LDH release and reduced MTT metabolism dose and time
dependently. A marked increase in LDH release was already detectable at
24 h after exposure. Since 5 h after staurosporine application the
production of mitochondrial reactive oxygen species by staurosporine
had initially been reported in chick embryonic neurons
(43)
, we also assessed lipid peroxidation in our model. At
this early time point the level of malondialdehyde transiently
increased from 0.116 ± 0.004 µM in vehicle-treated sister
cultures (n=10) to 0.224 ± 0.009 µM after treatment
with 300 nM staurosporine (n=8; P<0.001).
Pretreatment with 0.1 mM and 0.5 mM melatonin significantly reduced the
staurosporine-induced malondialdehyde formation to 0.159 ± 0.009
µM (n=6) and 0.165 ± 0.004 µM (n=10),
respectively (P<0.001 vs. 300 nM staurosporine). In spite
of this antioxidative effect, treatment with melatonin (0.1 or 0.5 mM)
did not prevent staurosporine-induced neuronal cell death. In
contrast, at the higher dose range melatonin appeared to aggravate LDH
release or loss of metabolic activity at all time points investigated
(Table 2
).
|
Effect of melatonin treatment on AF64A-induced neurodegeneration
Primary neuronal cell cultures from cortex were exposed to AF64A.
As reported previously, the neuronal cells reacted to AF64A with a
delayed dose- and time-dependent reduction in cell viability as
quantified by LDH release and MTT assay. At all doses of AF64A applied
(10–80 µM), LDH release was not detectable for up to 24 h. LDH
in the medium started to rise after 48 h, with the most pronounced
increase on day 3 of treatment (Fig. 3
; 44
). Likewise, even at the highest dose of AF64A, MTT metabolism was
not decreased before 16 h, but then gradually declined to reach a
minimum at 72 h after toxin application. Comparable results were
obtained when the exposure to AF64A was reduced to 5 h by removal
of the AF64A-containing medium and replacement by pooled preconditioned
medium (data not shown).
|
To differentiate whether the apoptotic neuronal cell death induced by
AF64A is caspase dependent or independent, the unspecific caspase
inhibitor Z-VAD-FMK was used. Treatment of cortical cells with 100 µM
Z-VAD-FMK before and during AF64A exposure completely prevented the
significant increase in LDH release occurring 48 h and 72 h
after AF64A application (Fig. 4
).
|
Pretreatment of cortical cultures with melatonin (0.1 and 0.5 mM 2 h before AF64A addition) did not attenuate the AF64A-induced LDH
release or prevent the reduction in MTT metabolism (Fig. 3
and
Fig. 5
). In contrast, AF64A-induced LDH release tended to be higher after
pretreatment with melatonin. Potentiation of the neurotoxic effect was
also evident at the low dose of AF64A (10 µM), when only a slight
increase in LDH release was detected (Fig. 3)
. Comparable results were
obtained when melatonin was added 1 h before AF64A (data not
shown).
|
To investigate whether oxidative stress plays a role in the
AF64A-induced neuronal damage and, if so, whether melatonin as a free
radical scavenger is able to avoid oxidative damage in this model, we
measured the extent of lipid peroxidation. In primary neuronal cultures
of cortex, the application of AF64A was followed by an increase in
malondialdehyde, which started after 10 h, was most pronounced
after 24 h, and was no longer detectable after 48 h. At this
time point apoptotic cell death was evident according to LDH release,
morphological changes, and DNA laddering (35
, 44)
. The
addition of melatonin (0.1 mM) 1 h before AF64A significantly
reduced the increase in malondialdehyde (Fig. 6
).
|
Effect of PBN and DMTU on AF64A- and staurosporine-induced
neurodegeneration
To obtain additional information on the involvement of free
radicals, two other general antioxidants, DMTU and the spin trap PBN,
were tested. Neither antioxidant prevented the increase in LDH release
72 h or 48 h after application of AF64A or staurosporine,
respectively (Table 3A
, B
). In contrast to melatonin, DMTU (1 or 10 mM) and PBN (0.1 or 1.0
mM) had no significant effect on the LDH release from native cells
within 72 h (Table 1)
. Comparable to melatonin, both PBN and DMTU
completely prevented the increase in malondialdehyde observed 24 h
or 5 h after addition of AF64A or staurosporine, respectively
(Table 3A
, 3B)
.
|
|
Effect of melatonin on OGD-induced neuronal cell death
Exposure of primary cultures of rat cortical neurons to OGD for
120 min resulted in a considerable increase in the release of LDH
within 24 h to an extent almost comparable to that induced by an
excess of glutamate (full kill). Melatonin dose-dependently reduced the
release of LDH. A significant reduction was achieved at a dose range of
0.5 mM, whereas at higher doses the neuroprotective effect vanished
(Fig. 7
).
|
On the other hand, neuroprotection by melatonin was not achieved when
OGD was performed in the presence of the NMDA receptor antagonist
MK-801, thereby blocking the excitotoxic component of hypoxia
(36)
. When cells were exposed to OGD for 2 h, MK-801
reduced the release of LDH to 40.6 ± 4.0% and 53.6 ± 3.7%
24 and 48 h after OGD, respectively. The protective effect of
MK-801 was considerably higher than that achieved by 0.5 mM melatonin
(reduction to 78.2±7.9% and 75.7±2.0, respectively; Table 4
). When OGD was prolonged to 3 h, MK-801 was less effective
(reduction of LDH release to 79.5±4.1%, both after 24 and 48 h)
and no further reduction was achieved by addition of melatonin (0.1 and
0.5 mM), whereas additional treatment with the caspase inhibitor
Z-VAD-FMK provided a further protection against OGD-induced neuronal
cell death (reduction of LDH release to 63.7±3.1% and 64.0±1.9%
after 24 and 48 h, respectively; Fig. 8
).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, OGD-induced cortical neuronal cell death was
prevented by melatonin at least in part. In contrast, no evidence was
found indicating the efficacy of melatonin in the case of apoptotic
neuronal cell death. This has been demonstrated in three different
models of apoptotic neuronal cell death. All three models depend on the
activation of caspases, as has been demonstrated previously
(36)
and/or in the present study, but differ in the
mechanisms and time course leading to apoptosis. In neuronal cell death
associated with apoptosis induced by AF64A or staurosporine, melatonin
was not neuroprotective. Moreover, a lack of neuroprotection by
melatonin was found in regard to the apoptotic component of OGD.
Neuronal cell death triggered by ischemia traditionally is regarded as
necrosis. Increasing evidence suggests that apoptosis may also
contribute to brain damage resulting from ischemic insults and the
apoptotic death can be unmasked with glutamate antagonists (36
, 45)
. In the present study, we demonstrated that melatonin was no
longer neuroprotective in OGD when the excitotoxic, NMDA
receptor-mediated hypoxic injury was prevented by treatment with the
glutamate antagonist MK-801 and, consequently, apoptotic cell death
prevailed. The limited effect of melatonin in hypoxia without blockade
of NMDA receptors (Table 3
and Fig. 7
) might be explained by the protective effect of melatonin restricted to
the necrotic component of ischemic cell death only. This is in
accordance with the observation that trolox, an antioxidant analog to
vitamin E, prevented necrosis but unmasked apoptosis in cultured
neurons subjected to oxygen deprivation (45)
.
Depending on the model for cell death, the variable efficacy of
melatonin requires the use of a variety of different models in order to
obtain a profound insight into the neuroprotective potential of this
compound. We therefore chose three models of apoptotic neuronal cell
death that differ considerably regarding the time course in which
neurodegeneration develops. The unique feature in the AF64A model is a
delayed onset of neuronal cell death in the cultures within 72 h,
with no morphological changes and no increase in LDH release 24 h
after exposure. In contrast, neuronal apoptosis induced by
staurosporine is fully visible within 24 h. OGD induced apoptosis
is fully developed after 24 h and the portion of total damage
remains stable for up to 48 h (Fig. 8)
. Previous experience
indicates that melatonin attenuates neuronal apoptosis in the case of
6-OHDA-, ß-amyloid-, and kainate-induced cell damage in
vitro and in vivo. 6-OHDA has been demonstrated to
trigger apoptosis in low concentrations in cultured dopaminergic
neurons, cerebellar granule neurons, and PC12 cells within 18–24 h
(46
47
48)
, whereas at higher concentrations of 6-OHDA cells
die by necrosis within 6 h (47)
. However, it has to
be considered that the mechanism by which 6-OHDA damages cells involves
the formation of hydrogen peroxide, superoxide, and free radicals
(49)
, which can be scavenged by melatonin. Oxygen free
radicals and increased sensitivity to excitotoxicity, which are
antagonized by melatonin, have been related to the cytotoxic properties
of ß-amyloid. The appearance of both apoptotic and necrotic cell
death has been described for ß-amyloid-induced toxicity depending on
the cell type (50
51
52)
. Moreover, the cytoprotective
properties of melatonin against ß-amyloid have been explained by the
in vitro ability of melatonin to modify the secondary
structure of ß-amyloid and to inhibit the formation of amyloid
fibrils (53)
. In vivo evidence is available
that melatonin protects against DNA damage, which was observed in the
hippocampus 48 and 72 h after intraperitoneal administration of
kainate to rats (54)
. The delayed onset of apoptotic cell
damage excludes a direct neurotoxic action of kainate and thus a direct
prevention of apoptosis by melatonin. More likely, the protective
effect against delayed apoptosis in response to kainate may have
resulted from the prevention of seizure activity by treatment with
melatonin (9
, 10
, 14)
. Kainate-induced apoptotic cell
death in hippocampal neurons also was prevented when seizures were
blocked by diazepam (55)
. The neuroprotective effect of
melatonin against kainate-induced brain damage is believed to be
related to its oxygen radical scavenging properties as well as its
antiepileptic and GABA receptor regulatory actions (10)
.
Moreover, after intrastriatal injection of kainate in the rat,
melatonin reduced delayed apoptotic neuronal death in adjacent cortex
but not in the striatum, again excluding a direct effect of melatonin
on apoptosis (56)
. Up-regulation of the glutathione
antioxidative defense system by melatonin has been suggested as a
mechanism for reducing neuronal death caused by excitotoxicity and for
preventing the kainate-induced damage from spreading to adjacent brain
regions (21
, 56)
.
In the present study, evidence has been obtained that melatonin
accelerates neuronal cell death in cultures. When added to native
primary neuronal cultures, melatonin per se decreased cell
viability as demonstrated by a significant increase in LDH within
72 h. This adverse effect of melatonin has not been documented
before. Either no values for control cultures treated with melatonin
were presented or the observation period was 24 h or shorter. At
this time point, however, no change in cell viability had yet appeared
in our experiments. No changes in LDH release within 72 h were
detected in response to PBN and DMTU. The slight cytotoxicity of
melatonin may indicate the presence of a prooxidant activity associated
with the antioxidant melatonin. This phenomenon has been described for
melatonin and other antioxidants including estrogen and vitamin C
(57
58
59
60)
. The increase in malondialdehyde induced by
melatonin also underlines the prooxidant potential of melatonin.
According to Reiter (3)
, melatonin is believed to work via
electron donation in detoxifying the · OH radical. In so doing,
melatonin must itself become a radical, the indolyl cation radical.
However, indole radical cations have been shown to react with oxygen,
giving rise to indoxyls via peroxy radical intermediates. The formation
of the radical cation of melatonin has been made responsible, e.g., for
the enhancement of vitamin E consumption induced by oxidative stress in
red blood cells (61)
. Medina-Navarro et al.
(60)
also suggested the formation of secondary oxidation
products as a possible mechanism for the prooxidant property of
melatonin. A prooxidant action would also explain our findings that
melatonin aggravated the AF64A- and staurosporine-induced LDH release,
and the protective effect in OGD started to vanish in the higher dose
range. Similarly, melatonin potentiated the neurotoxicity of
methamphetamine in rats (62)
. These findings, however,
would implicate that caution is necessary, when melatonin is used
therapeutically in higher concentrations in humans. In this respect,
the report of proconvulsant effects of oral melatonin in neurologically
disabled children is intriguing (63)
.
Various mechanisms are discussed by which melatonin mediates its
neuroprotection (see beginning of article). However, none of these
mechanisms was able to interfere with the progression of the
caspase-dependent apoptotic neuronal cell death in the three applied
models. This also refers to the antioxidant potential of melatonin.
Although the increase in malondialdehyde during the first 24 h
after application of AF64A was blocked by melatonin, this blockade did
not result in the avoidance of apoptotic cell loss. Thus, the presence
of oxidative stress in the AF64A model, which has been demonstrated
in vitro in the present study (Fig. 6)
and in
vivo after intracerebroventricular administration of AF64A
(64)
, does not appear to initiate and/or support the
progression of programmed cell death in this specific model. This has
been demonstrated in the staurosporine-induced apoptosis and may also
refer to the apoptotic component of OGD. The minor role of free
radicals in these models is further underscored by the inefficacy of
PBN and DMTU in preventing AF64A and staurosporine-induced cell death
in spite of the complete prevention of the formation of
malondialdehyde. In the case of excitotoxicity, melatonin prevented
both the increased formation of lipid peroxidation products and the
neuronal damage, as has been shown after hippocampal injection of
quinolinic acid (65)
.
In conclusion, the present data suggest that the neuroprotectiveness of melatonin strongly depends on the cause of neuronal cell death. As demonstrated in three different models of neuronal caspase-dependent apoptosis, the progression of apoptotic neuronal cell death was not inhibited by melatonin, in contrast to its effect on neuronal necrosis. If the formation of free radicals or the excess of excitotoxins are the major mechanisms responsible for the initiation of the cell death program, however, melatonin might be protective. In addition to this beneficial effect, evidence for the exaggeration of neuronal damage by melatonin has been obtained. Melatonin might be considered an endogenous neuroprotective factor useful for the pharmacological treatment of neurodegeneration produced by glutamate excitotoxicity and/or oxidative stress, such as brain ischemia or epilepsia. In contrast, a therapeutic potential of melatonin in neurodegeneration mainly associated with the apoptotic type of neuronal cell death is questioned.
|
ACKNOWLEDGMENTS |
|---|
Received for publication October 14, 1999.
Revision received March 2, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B. Biochem. Mol. Biol. Int. 37,1063-1070[Medline]
B activation. FASEB J 12,685-693This article has been cited by other articles:
|
|
 |
|
  C. Harms, K. Albrecht, U. Harms, K. Seidel, L. Hauck, T. Baldinger, D. Hubner, G. Kronenberg, J. An, K. Ruscher, et al. Phosphatidylinositol 3-Akt-Kinase-Dependent Phosphorylation of p21Waf1/Cip1 as a Novel Mechanism of Neuroprotection by Glucocorticoids J. Neurosci., April 25, 2007; 27(17): 4562 - 4571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. N. Anisimov Effects of Exogenous Melatonin--A Review Toxicol Pathol, October 1, 2003; 31(6): 589 - 603. [Abstract] [PDF]
|
|
|
|
 |
|
 T. WALTHER, L. OLAH, C. HARMS, B. MAUL, M. BADER, H. HORTNAGL, H.-P. SCHULTHEISS, and G. MIES Ischemic injury in experimental stroke depends on angiotensin II FASEB J, February 1, 2002; 16(2): 169 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Katchanov, C. Harms, K. Gertz, L. Hauck, C. Waeber, L. Hirt, J. Priller, R. von Harsdorf, W. Bruck, H. Hortnagl, et al. Mild Cerebral Ischemia Induces Loss of Cyclin-Dependent Kinase Inhibitors and Activation of Cell Cycle Machinery before Delayed Neuronal Cell Death J. Neurosci., July 15, 2001; 21(14): 5045 - 5053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Harms, M. Lautenschlager, A. Bergk, J. Katchanov, D. Freyer, K. Kapinya, U. Herwig, D. Megow, U. Dirnagl, J. R. Weber, et al. Differential Mechanisms of Neuroprotection by 17 {beta}-Estradiol in Apoptotic versus Necrotic Neurodegeneration J. Neurosci., April 15, 2001; 21(8): 2600 - 2609. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||
increase in LDH release as the difference from vehicle-treated sister
cultures. The LDH activity in vehicle-treated sister cultures was
61.2 ± 4.7 units/ml medium. (n=4–11 for each
condition, pooled from 2 different sets of experiments;
*P<0.001 vs. vehicle-treated cultures;
+P<0.005 vs. AF64A). 
Vehicle: (shaded bar) 0.1 mM
melatonin, 
0.5 mM melatonin.
