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1
* Cellular Neurobiology Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224, USA;
Department of Neurological Surgery and Program in Neuroscience, University of South Florida College of Medicine, Tampa, Florida 33612, USA; and
Department of Physiology, Nagoya City University Medical School, Nagoya 467, Japan
1Correspondence: Cellular Neurobiology Branch, National Institute on Drug Abuse, National Institutes of Health, 5500 Nathan Shock Dr., Baltimore, Maryland 21224, USA. E-mail: cborlong{at}intra.nida.nih.gov; or H.N, E-mail: hitoo-n{at}med.nagoya-cu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: stroke • gliosis • astrocytes • cell death • free radicals
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Astrocytes have been demonstrated
in vitro to possess receptors (3
, 4)
as well as
signaling molecules that can trigger neuronal messages that are key to
cell survival (5)
or death (6)
. Examination
of cell death has been primarily investigated using pure neuronal cell
cultures; when in vivo studies are conducted, models of
neurodegeneration have been evaluated mainly using neuronal death as
sole index. Of note, mixed astrocyte-neuronal cultures that resemble
the in vivo condition appear to promote better neuronal
survival than pure neuronal cultures (7
, 8)
.
The identification of trophic factors, such as glial cell-line derived
neurotrophic factor (GDNF) (9)
, has
prompted investigations into possible therapeutic actions of glial
cells. Glial cells are the main source of transforming growth factor
ß (in which GDNF is a subfamily member) and astrocytes have been
shown to release many growth factors under normal conditions or in
response to brain injury (10
, 11)
. Accordingly,
experimental treatment strategies for neurodegenerative disorders such
as Parkinson’s disease have exploited the support and trophic factor
properties of glial cells (12)
. For example, the
maturation of neurite axons and astroglial fibers from embryonic grafts
is characterized by donor glial cells remaining proximal to the grafted
site (13
, 14)
, thus allowing axons of passage to reach
host targets (15)
. Transplantation of astrocytes
transfected with the gene synthesizing and secreting GDNF or the
dopamine precursor L-DOPA have been demonstrated to provide enhanced
amelioration of parkinsonian symptoms (16)
. Grafted
embryonic dopaminergic neurons combined with infusion of astrocytic
growth factor or GDNF have similar positive effects.
Additional properties of astrocytes include their ability to control
water and to reduce glutamate toxicity (17
, 18)
.
Astrocytes siphon excess extracellular water and potassium ions, then
either redistribute them to their networks or excrete them into the
blood vessels. They also transport glutamate into soma and, in the
process, detoxicate glutamine by converting toxic
OH- into less harmful
H2O2. These findings
suggest that glial cells can exert protective effects on neuronal
survival by their trophic, siphoning, and detoxicating actions.
Cerebral ischemia has been associated with marked cell damage
characterized by widespread activation of glial cells or reactive
gliosis. There is disagreement as to whether such gliosis is a cell
death or a neuroprotective response. In experimental models of
ischemia, some studies have reported that astrocytes are more resistant
than neurons (19
, 20)
, but other investigations provide
equally compelling evidence of a higher vulnerability of astrocytes
than neurons (21
, 22)
. Because of the presence of dense
glial cell accumulations in the ischemic penumbra, their role in
propagating or limiting the size of infarction is widely debated
(11)
. Notwithstanding, the highly glial cell-populated
ischemic penumbra has been suggested to be a conducive target site for
cellular treatment intervention (23
, 24)
. Transplantation
of fetal (25
, 26)
or cultured neurons (27)
near or within the ischemic penumbra has been found to induce
behavioral recovery in ischemic animals. In the clinic, the ischemic
penumbra is also targeted by anticoagulants or thrombolytics to
dissolve blood clots (28)
. Although drug therapy remains
the treatment of choice for stroke patients, there has been no
conclusive evidence of long-lasting motor and cognitive improvement
with any of the current drugs (28)
. Thus, stroke remains
one of the main causes of death in the world and finding ways to rescue
the central nervous system after ischemia has been a major research
endeavor.
Recent studies have implicated the abnormal accumulation of free
radicals in neurodegenerative disorders. Free radical scavengers have
been shown to protect against cell death (29
, 30)
.
Melatonin is a highly potent free radical scavenger (31
, 32)
and its administration to rats has been found to be
effective against neurotoxicity (33
34
35)
. The present
study explored the utility of melatonin in ameliorating the deficits
associated with cerebral ischemia, which are in part mediated by
aberrant free radicals and/or reactive nitrogen and reactive oxygen
intermediates (e.g., production of nitric oxide, hydrogen peroxide,
peroxynitrite). Recent reports have demonstrated protective effects of
melatonin against experimental ischemic damage (29
, 36
37
38
39)
and a deficiency in melatonin has been suggested in
stroke patients (40)
. To date, the proposed mechanism for
the protective action of melatonin involves a direct free radical
scavenging effect on neurons. Because of obvious alterations in glial
cells after cerebral ischemia, we hypothesized that if melatonin
elicited therapeutic effects against cerebral ischemia, then it could
exert protective actions on glial cells, as well as the neurons. The
present results offer support for the emerging view that glial cells
are equally active participants as neurons in the maintenance of a
functional central nervous system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ischemia
Ischemia surgery has been reported elsewhere
(25
26
27)
. Briefly, rats were anesthetized by halothane and
an embolus (a 4–0 Ethicon nylon filament, with tip diameter tapered to
the size of a 26G needle) was inserted from the external carotid, via
the common carotid, to the base of the right middle cerebral artery
(MCA) to stop the blood flow to the MCA. During the ischemia,
anesthesia was cut off and animals were allowed to recover for 1 h; behavioral tests (see below) were performed to confirm successful
unilateral ischemia. After 1 h of ischemia, the animals were
reanesthetized with halothane; the embolus was withdrawn, the blood
reperfusion was established, and the skin was sutured. The body
temperature of animals was maintained at 37°C throughout the surgery
until they recovered from anesthesia.
Drugs
Melatonin (Sigma, St. Louis, Mo.) was dissolved in saline (using
a vortex) and administered orally (26 µmol/rat) just prior to 1 h MCA ischemia, then once daily for 11 or 19 consecutive days. Saline
was administered similarly to the other treatment group. Each animal
received an intragastric application (using an 18 gauge gavage) of 1 ml
of the solution.
Behavioral tests
For behavioral examination, the forelimb akinesia (also called
postural tail-hang test) and spontaneous rotational tests were
evaluated during MCA occlusion (i.e., during 1 h embolism) and
again at 11 and 19 days after ischemia-reperfusion injury. Detailed
description and specific criteria for each test to determine successful
ischemia have been reported elsewhere (27)
. Briefly, the
forelimb akinesia test involved holding the animal by the tail and
noting the positions of the forelimbs. Animals with MCA occlusion
demonstrate a characteristic posture with the left forelimb clipped to
their chest muscles while the right forelimb is stretched out. For the
spontaneous rotational test, the animal was placed in a box made of
transparent Plexiglas (40x40x35.5 cm) and the direction of the
animal’s rotation was noted over a five-minute session. Two full turns
(tight ipsiversive rotations toward the side of the ischemia) per
minute indicated complete MCA occlusion.
Immunohistochemistry
Detailed description of immunohistochemistry was reported
elsewhere (41
, 42)
. Briefly, animals under deep anesthesia
were perfusion-fixed transcardially with 4% paraformaldehyde at 11 or
19 days after ischemia. Brains were removed and cryosections (50 µM)
were processed for immunohistochemistry by a standard ABC method using
anti-GFAP antibody (Dakopotts, Roskilde, Denmark), anti-MAP2 antibody
(Chemicon, Temecula, Calif.), biotinylated rabbit anti-rat
immunoglobulin G (IgG; Vector Lab, Burlingame, Calif.), and
anti-aquaporin-4 antibody. Alternate sections were processed for
hematoxylin-eosin (H&E) staining. The extent of infarction was assessed
by measuring the maximum infarcted area (IgG extravasation) in each
animal using an NIH imaging system.
Astrocyte culture
The method for culturing primary astrocytes is described
elsewhere (43)
. Briefly, the cortical tissue from neonatal
rats was dissected and meninges were removed. The tissue was
mechanically dissociated and trypsinized at 37°C for 20 min. Trypsin
action was stopped by soybean trypsin inhibitor with DNase. The pellet
was suspended in complete medium (DMEM+10% FBS), plated on sterile
culture flask, and kept in an incubator with 5% CO (2)
at
37°C. After confluence, the culture flask was shaken to eliminate
cells other than astrocytes, then the cells were dissociated and seeded
in 24-well culture plates in complete medium. After confluence, the
medium was changed to DMEM/F12 without serum. Sodium nitroprusside
(SNP; a direct nitric oxide donor; 100 µM or 1 mM) or
3-ntiropropionic acid (3-NP; a succinate dehydrogenase inhibitor and
peroxynitrite donor; 1.7 mM) with or without melatonin (10 µg/ml)
were loaded to indicated wells.
MTT and neutral red assays
To investigate the protective effects of melatonin on astrocytes
against the cytotoxicity of 3-NP or cell death after serum deprivation,
pure astrocytes were cultured on 96-well dish at a density of
10,000/well (DMEM/F12+10% FBS). Treatment with 3-NP (6 mM) or serum
deprivation with or without melatonin (100 µM) was initiated at 3
days after plating. 3-(4,5-Dimethyl thiazoly) 2,5-diphenyl-tetrazolium
bromide (MTT) assay, which reflects the activity of succinate
dehydrogenase (Cell Counting Kit-8, Dojindo, Japan), was carried out 10
days after serum deprivation. Neutral red assay, instead of MTT, was
performed at 3 days after 3-NP treatment because 3-NP interferes with
the MTT assay by also inhibiting succinate dehydrogenase activity
(44
, 45)
. MTT assays were conducted in two independent
experiments (n=8 samples); neutral red assays involved three
independent experiments (n=12 samples).
Statistical analyses
Analyses of variance were used to reveal statistical
significance between the two treatment groups over the two test
periods. Since we found no significant differences in the behaviors or
histological disturbances in animals within group at both time periods,
we combined data from both time periods within group for subsequent
analyses. Scheffe’s t tests were used to compare the two
groups in the final analyses of the in vivo data, as well as
for analyzing the in vitro data. Differences were considered
significant at P < 0.05. Values are expressed as mean ± standard error (SE).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Melatonin markedly reduced infarctions
Histological analyses using H&E and IgG immunostaining of brains
revealed that ischemic animals treated with melatonin showed
significantly smaller infarcted areas than those of vehicle-treated
ischemic animals (Figs. 1
and
2
). This significant reduction of infarction by melatonin was
consistently noted at both days 11 and 19 after ischemia/reperfusion
injury. Both cortical and striatal infarcted areas were significantly
reduced in melatonin-treated animals, with the cortex displaying more
sensitivity to melatonin protection than the striatum. Using NIH
imaging, we measured the maximum infarcted area in serial sections of
the forebrain of each animal. Melatonin-treated animals exhibited
5.5 ± 4.5 mm2 (mean ± SE)
and 7.2 ± 4.0 mm2 whereas saline-treated
ischemic animals displayed 14.5 ± 2.0 mm2
and 10.6 ± 3.0 mm2 cortical and striatal
infarcted areas, respectively. Thus, melatonin decreased by ~60% and
30% the area of cortical and striatal infarction (Fig. 1E
).
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Melatonin partially blocked glial cell loss and gliosis produced by
ischemia
Histological disturbances in glial cells and neurons were
monitored using glial fibrillary acidic protein (GFAP) and microtubule
associated protein-2 (MAP2) immunostaining. Melatonin-treated animals
showed less glial cell loss, as well as gliosis (Fig. 2D
, 2E
) compared to vehicle-treated animals (Fig. 2A
, 2B
). Although both groups of animals displayed surviving
MAP2-positive pyramidal neurons in the cortex (Fig. 2C
, 2F
), glial cell support to these neurons was
detected in melatonin-treated animals, but not in vehicle-treated
animals. The reduction in glial cell loss and gliosis in
melatonin-treated animals were noted at 11 and 19 days
postischemia/reperfusion injury.
Interactions between glial cells and neurons in the ischemic brain
Three typical regions could be discerned after
ischemia-reperfusion injury in both striatum and cortex, which
reflected glial cell–neuron interactions (Fig. 3
). These areas included the ischemic core, where there was no detectable
survival of neurons and glial cells (Fig. 3A, B
, far
lateral), the ischemic penumbra, which was highly populated by glial
cells (Fig. 3B
, left end), and the zone between the ischemic
core and penumbra comprised of surviving neurons devoid of glial cells
(Fig. 3C
, D
, E
). For example, surviving pyramidal neurons were
noted next to the ischemic penumbra where there was a near absence of
astroglial support as revealed by no detectable GFAP-positive
astroglial cells (Fig. 3D
). Immediately adjacent to this
pure neuronal zone were areas of complete cell/tissue loss
(infarction). Interspaced between the pure neuronal zone and the area
of gliosis was a region containing surviving neurons that were
individually enveloped by glial cells exemplifying glial cell support
to neuronal survival (Fig. 3E
, 3F
). Thus,
the absence of glial cells in ischemic regions corresponded to areas
near the ischemic core, whereas their presence coincided with the
ischemic penumbra and areas near intact tissues. These distinct
ischemic areas were noted at 11 and 19 days postischemia/reperfusion
injury.
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Regional distribution of glial cells in response to ischemia
Because the cortex was primarily affected by melatonin, we further
investigated the distribution of glial cells in the remodeling of this
region after ischemia/reperfusion injury. In saline-treated ischemic
animals, migrating glial cells were detected at the edge of the lateral
ventricular zone of both hemispheres as revealed by immunostaining with
GFAP (a marker for astroglial cells) and aquaporin-4 (a marker for
water channel protein of astroglial cells) (Fig. 4A, B
). Regional analyses of the cortex revealed that
astroglial cells were almost undetectable and only neurons remained
close to the ischemic core (Fig. 4Aa
). At the edge of
cortical ischemic penumbra, astroglial cells exhibited dense
immunoreactivity (gliosis) characterized by several processes with
multiple branches/arbors and dark nuclei (Fig. 4Ab
). Similar
astroglial cells were noted near the edge of the ventricular zone of
the ischemic side, but their soma were stout and processes were not as
elaborate as those residing near the edge of the ischemic penumbra
(Fig. 4Ac
). Astroglial cells were also detected on the
contralateral cortex corresponding to ischemic penumbra (Fig. 4Ae
) and ventricular zone (Fig. 4Af
),
but with less elaborate processes and lightly stained nuclei. Normal
resident glial cells were detected in the contralateral intact cortex
corresponding to ischemic core (Fig. 4Ad
). Aquaporin-4
immunoreactivity resembled the GFAP pattern. Near total absence of
aquaporin-4 immunoreactivity, with only few lightly stained neuronal
somas, was seen close to the ischemic core (Fig. 4Bg
),
whereas in the corresponding contralateral intact side, normal
aquaporin-4 immunoreactivity was detected (Fig. 4Bj
). At the
edge of the ischemic penumbra, as well as in the contralateral
corresponding region, a strong aquaporin-4 immunoreactivty was detected
(Fig. 4Bh, k
). A denser aquaporin-4 immunoreactivity with
condensed glial soma was noted near the edge of the ventricular zone of
both hemispheres (Fig. 4i
). The gradient of GFAP and
aquaporin-4 immunoreactivity delineated distinct regions where viable
glial cells remained after the ischemia/reperfusion injury.
Specifically, the ventricular zone (gliogenesis site) and the edge of
the ischemic penumbra were heavily populated by glial cells. These
observations were consistent for both days 11 and 19 after ischemia.
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In vitro replication of melatonin protection against
ischemia
Ischemia/reperfusion damage entails nitric oxide or peroxynitrite
production, as well as interruption in cerebral blood flow, and these
cellular mechanisms were mimicked in vitro by treatments of
SNP or 3-NP and by serum deprivation, respectively. Pure astrocytic
cell cultures treated with melatonin (10 µg/ml) were protected
against cell death induced by SNP, 3-NP, or serum deprivation
(Fig. 5
). Treatment with SNP (1 mM or 100 µM) resulted in marked astrocytic
cell death at 36 h or 72 h, but cotreatment with melatonin
almost completely blocked the toxicity of SNP at both time periods.
Treatment with 3-NP (1.7 mM) also induced astrocytic cell death at
96 h, but cotreatment with melatonin (10 µg/ml) markedly reduced
the 3-NP toxicity. Melatonin also substituted for serum, as revealed by
survival of astrocytes after 10 days of serum deprivation. MTT and
neutral red assays further demonstrated that melatonin (100 µM) had
protective effects against 3-NP (6 mM) toxicity) (Fig. 6A
) and cell death after serum deprivation (Fig. 6B
). These protective effects of melatonin on pure astrocyte
cell cultures parallel the in vivo observations that
melatonin preserved the survival of glial cells after ischemic injury.
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Partial maintenance of normal motor functions by melatonin
All animals used in the present study were impaired in their motor
functions when examined during the 1 h occlusion of the right MCA.
The demonstration of asymmetrical behaviors during the occlusion period
in both melatonin- and saline-treated ischemic animals is indicative of
successful ischemic stroke (46)
, and such functional
deficits are stable for at least 6 months (27)
. When
evaluated at 11 and 19 days postischemia/reperfusion injury, the
majority of melatonin-treated ischemic animals displayed near normal
motor functions compared with saline-treated ischemic animals. In the
forelimb akinesia test, only 4 of 11 melatonin-treated animals
exhibited impairment compared to 8 of the saline-treated ischemic
animals. In the rotational test, only 1 of 11 melatonin-treated
ischemic animals displayed behavioral abnormality compared to 6 of 11
saline-treated ischemic animals. Thus, both behavioral tests revealed
that melatonin treatment ameliorated ischemia-induced behavioral
deficits.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 48)
. Three major findings were noted in the present study that implicated the critical role of glial cells in neurodegeneration after ischemic stroke and in neuroprotection rendered by melatonin. First, the disappearance of GFAP-positive glial cells, but with survival of neurons, within a few days after the ischemia/reperfusion injury indicates that glial cells were more vulnerable than neurons. One may argue that ischemia only induced some structural changes in astroglial cells that resulted in decreased antigenicity of GFAP protein, which would suggest that glial cell death did not precede neuronal death. However, the aquaporin-4 immunostaining (also a specific marker of astrocytes in the brain), conducted at the same time as that of GFAP, exhibited the same pattern as GFAP immunoreactivity, suggesting that glial cell loss or gliosis occurred prior to neuronal death.
The second observation is that there were differential alterations in
glial cells in ischemic brain areas. The cortical infarction was
reduced more significantly than that in the striatum after melatonin
treatment. The presence of higher number of glial cells in the ischemic
cortex than that of the ischemic striatum in animals that were treated
with melatonin might have amplified the cortical protection.
Experimental evidence points to regional differences in density and
viability of astrocytes. For example, the ratio of glial cells to
neurons is about one to one in the rat striatum (49)
, but
glial cells outnumber the neurons in the cortex of rat
(50)
or human (51)
, especially in the aging
brain (52)
. Because homotypic glial cells were shown to
provide better dendritic growth (53)
, the higher number of
cortical astrocytes than striatal astrocytes may account for better
protection of the cortex than the striatum from ischemia. The presence
of functional glial cells in specific areas of the brain in response to
brain injury may explain also the observed differential protection. In
the adult brain, stem cells remain in the subventricular zone of the
forebrain and the dentate gyrus. From the former, it has been reported
that the proliferating cells mainly develop into astrocytes, which then
migrate into other brain areas (54)
. It is possible that
an ischemic insult could activate an enhanced gliogenesis and
migration. In the ischemic striatum, reactive astrocytes have been
detected in the medial aspect as early as 4 days after
postischemia/reperfusion injury, but it required an extra 3 days for
them to reach the lateral aspect (55)
. This delay in the
migration of astrocytes to the lateral striatum might have adversely
contributed to fully protecting the striatum. Alternatively, cortical
cells may be more sensitive than striatal cells to the presence of
glial cells as revealed by better survival of fetal cortical cells than
fetal striatal cells when cultured in astroglial enhanced medium
(56)
. Taken together, these data may explain the
preferential protection of the cortex over the striatum.
The third major point of the present study is that the protection of glial cells afforded by melatonin is supported by behavioral and in vitro data. The reductions in glial cell loss and gliosis in melatonin-treated ischemic animals were paralleled by the observations of near normal motor functions in these animals. The ischemia-induced behavioral deficits seem to be mediated largely by a functional cortex because melatonin-treated ischemic animals had minimal cortical infarction compared to saline-treated ischemic animals. Even though these animals also displayed a reduction in total striatal infarction, the lateral aspect of the striatum was still clearly damaged, suggesting that protection of the cortex may be sufficient for normalization of motor behaviors. The absence of behavioral protection by melatonin during the 1 h occlusion indicates that the drug (administered once before the arterial occlusion) did not block the functional deficits associated with the acute ischemic insult caused by interruption of cerebral blood flow. It appears that melatonin was protective against secondary cell death processes.
The positive in vivo effects of melatonin were replicated
in vitro as revealed by continued survival of
melatonin-treated astrocytes after serum deprivation or exposure to
toxins (3-NP and SNP), which paralleled some in vivo
cellular events observed in response to ischemia/reperfusion injury.
Recent reports have demonstrated the efficacy of melatonin against
ischemic damage (29
, 36
, 37)
. Melatonin is an effective
free radical scavenger and indirect antioxidant (29
, 30
, 33
34
35
36
37
, 57
, 58)
. Hydroxyl radicals (generated by hydrogen peroxide via
the Fenton reaction) and peroxynitrite anions are scavenged by
melatonin (31
, 57)
. In addition, it blocks singlet
oxygen-induced toxicity (59)
. Lipid peroxidation in the
brain produced by intoxication of free radical generating agents is
also reduced by melatonin (35
, 60)
. These studies
demonstrate that melatonin directly protects neural tissue from free
radical toxicity. To date, however, these studies have not examined
alterations in glial cells after melatonin treatment. The possibility
exists that enhanced survival of glial cells after melatonin treatment
may confer protection to the injured neurons. The enveloping action of
glial cells on neurons might aid in the homeostasis of the neuronal
cell membrane by siphoning excess potassium or by enhancing water
handling capacity (Fig. 7
). In addition, glial cells may serve as cystine/glutamate antiporter
system that can prevent glutamate toxicity (3
, 4
, 61)
.
Finally, glial cells can secrete trophic factors, including GDNF, which
has recently been shown to protect against experimental cerebral
ischemia (62)
. Thus, the combined buffering action,
anti-glutamate toxicity transporter mechanism, and trophic
factor-secreting potential of glial cells make them efficacious
neuroprotective agents, and they could be recruited by melatonin to
combat ischemia/reperfusion injury.
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Are glial cells beneficial or detrimental to brain injury? There is
experimental evidence on both sides making conclusions difficult on the
role of glial cells in the central nervous system. For example, it has
been argued that after a brain insult, repair of the injury site may be
limited due to the nonpermissive condition elicited by reactive gliosis
(63)
. In response to injury, astrocytes or microglia
secrete substances, such as basic fibroblast growth factor,
transforming growth factor ß or interleukins, which have been
demonstrated to exacerbate the degeneration of the injured site
(64)
. On the other hand, the same reactive gliosis has
been shown to promote reparative effects in the injured brain. In
response to excitotoxic lesions (65)
or intraparenchymal
infusion of basic fibroblast growth factor (66)
, reactive
astrocytes are involved in removing degenerating axon terminals, but
not axons of passage, from the neuropil. We have shown also that
ongoing glial response may underlie the long-term functional recovery
in transplanted parkinsonian rats (67)
. The present
results suggest that glial cells may participate in neuroprotection.
However, we do not exclude the equally beneficial effects of
exploiting protective or reparative strategies designed at
directly manipulating neuronal survival. Rather, we suggest that a
complete glial cell–neuron interaction model should be considered when
studying the central nervous system during states of homeostasis and
vulnerability.
From the basic research standpoint, the present results support the
notion that glial cells are closely involved in brain injury. We now
propose a dynamic interaction between glial cells and neurons during
ischemia/reperfusion cell damage (Fig. 8
). The first stage consists of initial activation of glial cells and
occurs within a few days after the ischemia/reperfusion process. The
second stage, about 1 wk later, is characterized by intense glial cell
loss that corresponds to the formation of the ischemic core. Surviving
neurons enveloped by the remaining glial cells, both with pruned
processes, can still be detected within the ischemic core. After total
disappearance of glial cell support, the third stage ensues with the
onset of infarction. It is critical therefore that treatment
modalities, such as the present melatonin application, should be
initiated during the period when glial cells are still present. Perhaps
the limited efficacy of drug therapy may be due to the short period of
time that glial cells can be rescued from cell death. Because injured
but viable glial cells are found along the ischemic penumbra, immediate
rescue of this region may promote better protection. Our better
understanding of glial cells, including their narrow window of
reversible injury and their location or migration pattern in the
ischemic penumbra, can greatly optimize the treatment for stroke.
|
In summary, we demonstrate alterations in glial cells during a period of neuronal vulnerability to undergo cell death after cerebral ischemia. Melatonin treatment exerted neuroprotection against ischemia/reperfusion injury by maintaining survival of both glial cells and neurons. The present study highlights the potential to protect the central nervous system from stroke by rescuing glial cells in addition to neurons. However, glial cells appeared more vulnerable than neurons after ischemia/reperfusion injury and also more sensitive to neuroprotective treatment. Therapeutic agents directed at glial cell protection may provide clinical applications for treatment of stroke and neurodegenerative disorders. Partial protection against cerebral ischemia was established by melatonin and perhaps enhanced recovery can be achieved by a combination of therapies designed at maximizing glial cell–neuron interaction.
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ACKNOWLEDGMENTS |
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Received for publication July 12, 1999.
Revision received February 29, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  R. J. Reiter, D.-x. Tan, J. Leon, U. Kilic, and E. Kilic When Melatonin Gets on Your Nerves: Its Beneficial Actions in Experimental Models of Stroke Experimental Biology and Medicine, February 1, 2005; 230(2): 104 - 117. [Abstract] [Full Text] [PDF]
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