Neuroscience & Medicine, 2010, 1, 50-59
doi:10.4236/nm.2010.12008 Published Online December 2010 (http://www.SciRP.org/journal/nm)
Added after Anoxia-Reoxigenation Stress,
Genistein Rescues from Death the Rat Embryo
Cortical Neurons
Carmen Arce 1, José Luis Arteaga 1,Eduardo Sánchez-Mendoza 1, María Jesús Oset-Gasque 1,
Sixta Cañadas 1, María Pilar González 1,2
1Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Universidad Complutense, Madrid, Spain; 2Centro de
Investigaciones Biológicas, CSIC, Ramiro de Maeztu, Madrid, Spain
Received September 7th, 2010; revised September 17th, 2010; accepted November 8th, 2010
ABSTRACT
Estrogens and phytoestrogens have neuroprotective effect against neuronal damage induced by cerebral ischemia
/reperfusion (I/R) injury. In preceding studies, the phytoestrogen effects have been assessed by administration previous
to the ischemic period, conditions which are unusual to apply to the treatment of human stroke. Here we present a study
on neuroprotection afforded by genistein on rat embryo cortical neurons subjected to oxygen and glucose deprivation
(OGD) followed by re-oxigenation, when added after the stress stimulus. At 1 and 2 h of OGD times and after 24 h of
reperfusion, cell viability, necrotic, apoptotic and autophagic cell death and different parameters related to oxidative
stress and mitochondrial dysfunction were measured in the absence and presence of 1 µM genisteine. We found an in-
creasing loss of neuronal viability after 1-5 h of OGD which was only reversed in part by 24 h of reperfusion. These
changes were preceded by increases in ROS generation, caspase-3 activation, LDH release and increase in LC3B lipi-
dation, indicative of autophagia. Treatment with 1 µM genistein during the 24 h reperfusion significantly attenuated
neuronal necrosis and autophagia induced by 1 and 2 h of OGD exposure. Genistein also decreased ROS generation
and lipid-peroxidation induced by 2 h of OGD. These results suggest an important neuroprotective effect of genistein
against transient post-isch emic-like conditions.
Keywords: Cortical Neurons, Oxygen-glucose Deprivation, Brain Ischemia, Neuronal Death, Necrosis, Apoptosis, Autophagy,
Neuroprotection, N eurorepair,Phytoestrogens, Genistei n
1. Introduction
Global cerebral ischemia is a clinical outcome that occurs
as consequence of different conditions like cardiac arrest,
coronary artery bypass surgery and reversible severe hy-
pertension. All these events are the cause of a dra matically
reduced blood supply of oxygen and nutrients, as glucose,
which are the responsible for cellular damage. Studies
performed during the last 20 years have identified several
biochemical and cellular events that lead to ischemic neu-
ronal degeneration [1,2]. Although the management of
stroke has improved remarkably over the last decade due
mainly to the use of thrombolysis, moreover, nowadays
there is a growing interest to develop treatments aimed to
promote repair and regeneration of brain tissue damaged
by ischemia, which could be more effective than neuro-
protection on the brain recovery after ischemia.
Our increasing knowledge concerning with the ischemic
cascade is leading to a considerable development of phar-
macological tools, suggesting that each step of this cascade
might be a target for cytoprotection. Thus, many neuro-
protective drugs, such as calcium channel blockers, anti-
oxidants or free radical scavengers, GABA agonists, glu-
tamate antagonists, growth factors, NO inhibitors, phos-
phatidylcholine precursors and so on, have been studied in
experimental stroke models (for review, see [3]). However,
very few have shown efficacy in clinical trials. Among
them, caspase inhibitors to reduce apoptosis and estrogen
or its derivatives, phytoestrogens, have been proposed as
future neuroprotecti ve treatm ent [4,5] .
In vivo and in vitro studies have demonstrated powerful
neuroprotective effects of estrogens against a variety of
insults. In rodents, estrogen reduces injury caused by focal
cerebral ischemia [6] and global ischemia [7]. In vitro,

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 51
similar neuroprotective effects have been demonstrated in
primary cortical [8-10], hippocampal [11-13] and mesen-
cephalic cultures [14]. Furthermore, these neuroprotective
effects appear to utilize multiple mechanisms depending
on the injury and cell type [5,15] .
It is increasingly clear that physiological doses of
isoflavones, which can behave as phytoestrogens, can
mimic some of the neuroprotective effects of estrogens.
[16-18]. In vitro some soy isoflavones can protect pri-
mary neurons from glutamate toxicity [19], thapsigar-
gin-induced apoptosis [20], and -amyloid toxicity [21].
Several studies have indicated that genistein admini-
stration prevents delayed neuronal death after transient
global cerebral ischemia [22], inhibits the lipid peroxi-
dation induced by pro-oxidant agents in cultured corti-
cal neurons [23] and attenuates the oxidative stress and
neuronal damage following cerebral ischemia in rat
hippocampus [24]. However, the mechanisms underly-
ing protection from ischemic injury remain unclear.
Recently, Schreihofera and Redmond, [17] have shown
that pre-treatment with dietary levels of soy phytoes-
trogens can mimic neuroprotective effects observed
with estrogen. Nevertheless, in all these work s estrogen
and phytoestrogens have been studied as neuroprotec-
tive agents but it has not been studied their possible
effects on neurorepair in post-ischemic period, in spite
that it is in this time when protection is necessary after
an ischemical event. The present study was performed
to test the ability of the phytoestrogen genistein to in-
hibit neuronal death in the post-ischemic period and to
obtain further evidence about the molecular mecha-
nisms by which phytoestrogens exercise these potential
neuroprotective and neurorepairing effects. The study
has been performed by using cortical neurons in pri-
mary cultures subjected to oxygen-glucose deprivation
(OGD), since this has been proved to be a good model
to test the mechanisms of neuroprotective agents in
brain ischemia. Our results suggest that genistein may
protect neurons from OGD in the post-ischemic period
by attenuating oxidative stress, lipid peroxidation, and
necrotic and autophagic cell death.
2. Material and Methods
2.1. Materials
Tetramethylrhodamine methyl ester (TMRM) and 2,7
dichlorodihydro-fluorescein diacetate (2,7 DCF-DA)
and bis-[1,3-diethyl-thio-barbiturate]-trimethine-oxonol
(bisoxonol) were purchased b y Molecular Probes ( E u ge n e ,
OR). Minimum Essential Eagle’s Medium (EMEM)
was from Bio-Whittaker, and Foetal Calf (FCS) was
from Sera-Lab (Sussex, England). Other chemicals
were reactive grade products from Merck (Darmstadt,
Germany). Anti-rabit microtubule associated protein
light chain 3 (LC3B) antibody was from Cell Signal-
lyng Technology Inc. (Danvers, MA).
2.2. Cell Isolation and Culture
Foetal rat brains of 19 days gestation were used. Brain
neurons were obtained as described in [25]. Isolated
neurons were suspended in EMEM medium containing
0.3 g/l glutamine, 3 g/l glucose, 10% FCS, 100 U/ml
penicillin and 100 µg/ml streptomycin. Cells, at density
of 106 cells/cm/well, were placed on plastic Petri dishes,
treated with 10 µg/ml poly-D-lysine, so that the cells
could attach themselves to these plates. Incubations
were made in a humidified incubator with 5% CO2/95%
air at 37˚C. After 72 hours, the incubation medium was
replaced by fresh medium to which 10 µM of cytosine
arabinoside was added to prevent overgrowth of con-
taminating glial cells. Cells were used after 7 days of
culture. Cell purity was checked by both, cells staining
with cresyl violet to identify neurons, and immunocy-
tochemistry with the specific anti-GFAP antibody [25]
to identify glial cells. Under these conditions, the glial
cell number in the cultures was of 8.3 ± 3.6% of the
total cell population (neurons + glial cells).
2.3. Oxygen-glucose Deprivation (OGD)
After 7 days of culture, the medium was removed and
replaced by a glucose-free balanced salt Locke medium
(composition (mM) NaCl 140, KCl 4.7, KH2PO4 1.2,
CaCl2 2.5, MgSO4 1.2 and HEPES 15, pH 7.5). Multi-
wells were then placed in a gassed chamber equipped
with gas entry and outflow devices, the chamber was
then sealed and placed in the gassed incubator in an
atmosphere of 95% N2/5% CO2 at 37˚C for 1 or 2 h.
OGD was terminated by removing the cultures from the
chamber. These cells were considered as cells treated
with OGD. In some multiwells, after this treatment, the
OGD cultured medium was replaced by normal medium
(EMEM containing 3 g/l glucose) and the multiwells
were returned to the incubator under normoxic condi-
tions (95% air/5% CO2). This treatment was considered
as reperfusion. The time of reperfusion of OGD-neurons
was of 24 h. Control experiments were performed al-
ways in parallel, so, after 7 days of culture, the medium
was removed and replaced by fresh normal medium
EMEM containing 3 g/l of glucose. Control cells were
kept at incubator under this condition during 1 or 2 h.
Then, OGD-neurons were processed as indicated in
each case.
2.4. Assessment of Cell Viability
Cell viability was determined by the XTT test, based on
the ability of living metabolically active cells to reduce

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 52
the yellow tetrazolium salt (XTT) to form an orange
formazan dye, which quantity directly correlates to the
number of living cells. Measurements were preformed as
described [25]. Briefly, control and treated neurones (2 ×
105) were incubated with the XTT solution at 0.3 mg/ml
final concentration and the orange formazan dye formed
was spectrophotometrically quantified using an ELISA
plate reader , at 450 nm (reference 650 nm). Results were
expressed as percentages with respect to the control cells.
2.5. Measurement of the ROS Formation
To ass ay the ROS for mat i on, 2,7-dic hlorodihydro fluorescein
diacetate (H2DCF-DA), a non-fluorescent lipophilic re-
agent, was used. HDCF-DA en ters in to the cells, wh ere it
is transformed into 2,7-dichloro dihydrofluorescein (H2DCF)
by the action of intracellular estearases. H2DCF is oxi-
dized to fluorescent DCF by hydrogen peroxide. H2DCF
-DA (5 M) was added to the cells, before subjectting
them to the different treatments and then the incubation
medium was removed, the cells were washed twice with
PBS and the fluorescence was measured in a FL600-Bio
Tek spectrofluorimeter with filters of 485/20 nm exc and
530/25 nm em. Results were expressed as arbitrary fluo-
rencence units (AFU) ratios.
2.6. Lactate Dehydrogenase (LDH)
LDH activity was measured as the rate of decrease of the
absorbance at 340 nm, resulting from the oxidation of
NADH to NAD+ as described [25]. Briefly, the culture
medium was collected and the neurones were lysed with
0.1 M Tris-HCl (pH 7.4) containing 0.1% Triton X-100,
cell suspension was centrifuged at 13,000 g during 5
minutes and LDH activity measur ed in both prep arations,
culture medium and cells supernatant. Activity of the
LDH release is given as percentage of release of LDH
with respect to the total LDH conten t (LDH in the cultu re
medium plus LDH inside the cells).
2.7. Caspase-3 Activity Measurement
Control and treated cortical neurones were washed rap-
idly with PBS and lysed by cell lysis buffer (10 mM
Tris-HCl, 10 mM NaH2PO4/Na2HPO4, pH 7.5, 130 mM
NaCl, 0.5% Triton X-100, 10 mM Na4P2O7). Lysates
were centrifuged at 13,000 g for 5 minutes and cas-
pase-3 activity was measured in the supernatants. Su-
pernatants with at least 20 g of protein were incubated
in caspase-3 assay buffer (20 mM HEPES, pH 7.5, 10%
glycerol, 2 mM DTT) , con tain ing 20 M Ac. DEVD-AMC.
[N-acetyl-Asp-Glu-Val-Asp-(7-amino-4-methylcoumarin) at
37˚C during 2-4 h. The fluorogenic AMC liberated from
Ac-DEVD-AMC was monitored using a spectro fluori-
meter (Bio-Tek FL 600) with an excitation wavelength of
360/20 nm and an emission wavelength range 460/20 nm.
Under these conditions, the caspase-3 activity was linear
during 6 h. Enzymatic activity is expressed as arbitrary
fluorescence units, after 3 hours, per g protein (AFU/3
h/g protein).
The cell protein contents were monitored by the Brad-
ford technique [26].
2.8. Mitochondrial Membrane Potential Assay
Mitochondrial membrane potential was measured ac-
cording to Lalitha Tenneti et al. [27], with few modifica-
tions. Control and treated cells were washed with PBS
and incubated for 30 min with 500 nM of TMRM dis-
solved in Locke medium (140 mM NaCl, 4.4 mM KCl,
2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4 mM
NaHCO3, 5.5 mM glucose and 10 mM HEPES, adjusted
to pH 7.5). Then, the cells were washed with PBS, and
the fluorescence was measured with a FL600-BioTek
spectrofluorimeter using filters of 530/25 nm exc. and
590/35 nm em.
2.9. Plasma Membrane Potential Measurement
Changes in the membrane potential (PMP) of neurons
were monitored with the fluorescent dye bisoxonol (bis-
[1,3-diethyl-thio-barbiturate]-trimethineoxono l), which is
a lipophylic anion whose distribution across the mem-
brane is dependent upon the membrane potential. Thus,
an increase in bisoxonol fluorescence indicates that the
membrane has been depolarised, allowing more of this
negatively charged dye to enter the cells [28]. The con-
trol neurons, and neurons after treatment were washed
and incubated with 0.2 M bisoxonol for 30 min. After
that, bisoxonol was removed, the cells were washed with
PBS and suspended in PBS and fluorescence was meas-
ured at wavelengths of 540 nm excitation and 565 nm
emission, and monitored with a FL600-BioTek spectro-
fluorimeter. Fluorescence intensity was reported in arbi-
trary fluorescence units (AFU).
2.10. ATP Determination
ATP analysis was performed by using the luciferase re-
action. Cells were lysed with 0.4 M perchloric acid and
the lysates neutralized with 1 M KOH under ice. The
cellular extract was centrifuged and supernatant were
kept at 0˚C. Aliquot samples were assayed immediately
with firefly luciferase/D-luciferin as indicated in the test
protocol (ATP Bio-Orbit Kit) and 2 mM of EDTA in 0.1
M Tris-acetate buffer, pH 7.5. The increase in chemilu-
miniscence was recorded in a Bio Orbit 125 luminometer.
For calibrating the light signals, 10 pmol of ATP dis-
solved in Tris-acetic buffer was injected into each sample,
and luminescence signal was determined. ATP cellular
content was expressed as arbitrary fluorescence units
(AFU). Intracellular ATP content in basal conditions was

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 53
of 670 ± 32 pmol ATP/106 cells.
2.11. Measurement of Lipid Peroxidation
The membrane lipid peroxidation in neurons was assayed
according to Fraga et al. [29]. After OGD treatment of
neurons, the culture medium was removed and cells were
washed twice with PBS, and 0.1 ml of ethanol solution of
butylated hydroxytoluene (4%), 0.5 ml of SDS (3%) and
0.3 ml of phosphotungstic acid (10%) was added to the
neurons. The suspension was shaken and incubated at
100˚C for 45 min, and then 1 ml of 2-thiobarbituric acid
(0.7 %) and 2 ml of HCl (0.1 N) were added. Once the
suspension was cooled, 3 ml of n-butanol was added,
shaken, and centrifuged at 2,000 g for 10 min. Then, the
cells were washed with PBS, and the fluorescence was
measured in the butanol phase with a FL600-BioTek
spectrofluorimeter using filters of 515 nm exc. and 555
nm em.
2.12. Western Blot
Cortical neurons were plated on 12 well plates and treated
as indicated in oxygen-glucose-deprivation section. Cells
were washed, harvested, and lysed and western blot was
performed as described by Figueroa et al. [25]. Rabbit
LC3B (1/2000) was used as primary antibody, and rabbit
peroxidase-conjugated IgG (1/2000) was used as secon-
dary antibody. The an tibody-reactive bands were revealed
by enhanced chemiluminiscence (ECL western detection
kit from Sant a Cruz Bi ot echn ology , I nc).
2.13. Data Analysis
Data are presented as means  SEM of three or four
separated experiments from different batches of cells
from different cell cultures, each one performed in tripli-
cated. Statistical comparisons were made using the Stu-
dent’s t test. This is a parametric test which studies
differences between two populations. Results are means
 SEM of three separate experiments, from cells of dif-
ferent cultures, each one performed in triplicate. Values
of p < 0.05 were considered as statistical significant. (*)
= p < 0.05, (**) = p < 0.01 and (***) = p < 0.001.
3. Results
3.1. Genistein Reduces the Loss of Neuronal
Viability Induced by OGD
As observed in Figure 1(a), exposure of cortical neurons
to OGD induced a loss in cell viability which was propor-
tional to the OGD time. Exposure of cortical neurons to 1h
OGD induced a loss in cell viability of about 40%, effect
which was significantly reversed by 24 h of reperfusion
(Figure 1(b)) but without reaching t he control val ue.
(a)
(b)
Figure 1. Effect of oxygen glucose deprivation (OGD) on
cell viability. (a) Effect of OGD exposure times on cell vi-
ability; (b) Effect of 24 h of reperfusion after 1 h OGD, in
the absence or presence of genistein, on cortical neuron
viability.
The addition of 1 M genistein during the 24 h post-
ischemic period, at the beginning of reperfusion, in-
creased the neuroprotective effect induced by reperfusion
alone and almost completely reverted the loss in cell vi-
ability induced by OGD (Figure 1(b)).
3.2. Genistein Attenuates the Necrotic Cell Death
Induced by OGD
As a loss in cell viability is indicative of cellular death,
necrosis, apoptosis and autophagy were studied. Results
from Figure 2(a) show that OGD induced an increase in
LDH release which is dependent on the cells exposure
time to OGD. The LDH increase, after 2 h of OGD
treatment, coincides with the loss in cell viability at this
time (about 50% in both cases). After 2 h of OGD, a pe-
riod of 24 h of reperfusion did not stop the LDH release
although it was lower than during OGD treatment (Fig-
ure 2(b)). This means that during the OGD treatment the
neurons have suffered a cellular membrane damage
which is impossibly to repair during reperfusion. How-
ever, the presence of genistein during reperfusion re-
versed the necrotic death to a great extend (Fi gure 2(b)).

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 54
(a)
(b)
Figure 2. Effect of OGD on LDH release. (a) Effect of OGD
time on LDH release; (b) Effect of 24 h of reperfusion after
2 h OGD, in the absence and presence of 1 M genistein on
LDH secretion.
3.3. Genistein Protects Againts ATP Lost
Since it is known that necrosis is associated with a loss in
intracellular ATP, the intracellular levels of this parame-
ter were studied. Results from Figure 3 show that after 2
h of OGD there was a great reduction in the intracellular
ATP levels which is in accordance with the cellular death
produced by necrosis (see Figure 2(a)). During reperfu-
sion the ATP levels continuously decay although at lower
levels than in the case of the OGD condition alone (Fig-
ure 3). However, when reperfusion was performed in the
presence of 1 M genistein, a light but significant in-
crease in the ATP content was observed with respect to
that in the absence of genistein (Figure 3).
3.4. Genistein Protects Cortical Neurons against
ROS Generation Induced by OGD
As the ROS formation could be an inductor of death by
necrosis, this parameter was also measured. Results from
Figure 4(a) indicate that the ROS generation is depend-
ent on time of cells exposure to OGD. During the 24 h of
reperfusion, after 2 h of OGD, ROS levels were signifi-
cantly reduced in about a 30% with respect to the ROS
induced for OGD. The treatment with 1 M genistein,
during the 24 h reperfusion period after OGD, induced a
total reversion of the ROS levels induced by the OGD
treatment (Figure 4(b)).
Figure 3. Effects of OGD and reperfusion, in absence and
presence of 1 µM genistein, on intracellular levels of ATP.
(a)
(b)
Figure 4. Effect of OGD on ROS formation. (a ) Effect of OGD
exposure times o n ROS formatio n; (b) Effect of 24 h reperfusion,
in absence or pre sence of genis tein, after 2 h of OGD.
3.5. Genistein Protects Neurons against Lipid
Peroxidation Induced by OGD
It is known that ROS may produce lipid peroxidation,
being this process the responsible for the necrotic cell
death. We measured this parameter under the OGD con-
ditions and the results showed an increase in lipid per-
oxidation after 2 h of OGD (Figure 5). During the 24 h
of reperfusion, after 2 h of OGD, lipid peroxidation lev-
els were significantly reduced in about a 65% with re-
spect to that induced by OGD, although they were higher

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 55
than in the control. The treatment with 1 M genistein,
during the 24 h reperfusion period after 2 h of OGD treat-
ment, produced a total reversion of the increase in lipid
peroxidation induced by the OGD treatment (Figure 5).
3.6. Genistein Doest not Protect Neurons against
Apoptotic Cell Death Induced by OGD
Knowing that in this OGD model neurons die by necrosis,
the possibility of an apoptotic cell death was also checked
and caspase-3 activity was measured as indicator of this
process. In our OGD model the cells were cultured in
absence of serum, then, as the serum deprivation induces
apoptosis in cortical neurons [25] a positive control of
apoptosis like the absenc e of serum (trophic factors) was
used. Results from Figure 6(a), indicate that the only
serum deprivation was able to induce an increase in cas-
pase-3 activation, being this effect directly proportional
to the time the cells were cultured without serum. On the
other hand, when these cells were subjected to OGD for
different times, the caspase-3 activity was also signifi-
cantly increased with respect to the treatment times.
These effects were higher than those found for neurons
cultured in absence of serum. Thus, the OGD treatment
“perse” was also able to induce additional increases in
caspase-3 activation (Figure 6(a)), although, at the low
OGD treatment (1 to 5 h), the increase in caspase-3 ac-
tivity with respect to the cells cultured in absence of se-
rum was lower than those produced at the longer OGD
conditions (24 h). The increase in caspase-3 activity in-
duced by 2 h O GD was significan tly reduced during 24 h
of reperfusion and, in this case, 1 M genisteine had not
any additional effect (Figure 6(b)).
3.7. Genistein Protects Mitochondrial Membrane
Potential without Affecting Plasma
Membrane Potential
One of the mechanisms of apoptosis cell death induction
is mitochondria malfunction. In order to check the in
Figure 5. Effects of OGD and reperfusion on lipid peroxidation,
in the absence and presence of 1 M g enist ein .
(a)
(b)
Figure 6. Caspase-3 activation in cortical neurons exposed
to OGD. (a) (-OGD) = action of time serum deprivation on
cortical neurons in culture. (+OGD) = Effect of different
OGD exposure times on caspase-3 activity. The activity of
caspase-3 at 0 time (control) was 5.9 ± 0.4. (b) Effects of 24
h of reperfusion after 2 h of OGD exposure in absence and
presence of 1 µM genistein.
volvement of mitochondria in apoptotic neuronal death
induced by OGD, the possible variations in mitochondrial
membr ane pot entia l (MMP) ind uced by the OGD trea tment
were checked. Results from Figure 7(a) show that 1 h of
OGD produ ces a sign ific ant in crea se in MMP. On the other
hand, 24 h of reperfusion in absence of genistein, after 1 h
of OGD, decr eased the TMR sign al wi th r espect to the con-
trol. However, when the reperfusion is performed in pres-
ence of 1 M genistein the MMP reach the values of the
control cells. By contrast with plasma membrane potential
(PMP), 1 h of OGD tr eatment did not aff ect th e potent ial of
the cellular membrane, in this case genistein had not any
effect on this parameter (Figure 7(b)).
3.8. Genistein Attenuates the Autophagic Cell
Death Induced by OGD and Reperfusion
It is known that neurons can undergo more than one type
of programmed cell death (PCD), not only apoptosis (or
type I PCD) but also autophagy (or type II PCD). Auto-
phagy was initially described as a process in which cyto-
plasmic material is degraded in lysosomes, providing
nutrients for survival. However, autophagic features have
also been observed in cells exposed to pathological in-
sults as well as in remodelling tissues [30,31]. Because

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 56
Figure 7. Effect of OGD and reperfusion in absence and
presence of 1 µM genistein on mitochondrial (MMP) and
cellular membrane (PMP) potential [A]: Action on MMP [B]
Effect on PMP.
several recent studies have shown that autophagy is in-
volved in ischemic brain damage [32,33] and nutrient
deprivation has been shown to lead cell to autophagia,
we check in this paper the possibility that in the OGD
experimental ischemia model used here, cortical neurons
could drive to enter in an autophagic mechanism.
To characterize autophagy, we carried out the assay of
LC3B lipidation (increase in LC3B-II from LC3BI)
measured by western blot. As it could be appreciated in
Figure 8, OGD was able to induce a significant increase
in LC3B-I lipidation to the autophagosomal LC3B-II in
cortical neurons. A utophagic cell death induced by OGD
was kept among 1 to 5 h of treatment, although the
maximum autophagia levels were observed at 1 h of
OGD treatment. At higher OGD exposure 2 and 5 h, the
autophagia was lower than at 1 h exposure but the values
were higher than in control cells. 24 h of reperfusion re-
versed in part this parameter. However, the reperfusion
in presence of 1 M of genistein completely reversed
LC3B lipidation.
4. Discussion and Conclusions
Epidemiologic data show that populations which con-
sume diets rich in phytoestrogens have low incidence in
coronary artery diseases [34,35]. Several studies have
indicated that genistein administration prevents delayed
Figure 8. Time-course effects of OGD and reperfusion, in
absence and presence of 1 M genistein, on LC3B lipidation.
Western blot analysis of LC3BI and LC3BII was performed
in extracts of cortical neurons subjected to OGD exposure
and 24 h reperfusion, in the absence or presence of 1 M
genistein, at indicate times, using a LC3B antibody. (a)
Representative LC3B western blot is shown. (b) Data are
expressed as ratios over basal control and are mean ± SEM
of two experiments each one performed in duplicate.
neuronal death after transient global cerebral ischemia
[22,36,37] and attenuates oxidative stress and neuronal
damage following cerebral ischemia in rat hippocampus
[24] and inhibits the lipid peroxidation induced by
pro-oxidant agents in cultured cortical neurons [23].
However, in all these works phytoestrogens have been
studied as neuroprotective agents when they are admin-
istered in the period previous to ischemia and not in the
postischemic period. Nevertheless it is in this time when
protection is necessary after an ischemic event. Therefore,
the present study was performed to test the ability of the
phytoestrogen genistein to inhibit neuronal death in the
post-ischemic period.
In the OGD model described here we found that there
was a loss in cell viability which was accompanied by
necrotic and apoptotic cell death, and we demonstrated
that both death processes are time-dependent. The exis-
tence of a necrotic cortical neuron death, induced by the
OGD model, is supported by the decrease in the intracel-
lular ATP levels, the great ROS generation and the pro-
duction of lipid peroxidation. This is in accordance with
Traystman et al. [38] and Matsuo et al. [39] who found
that a great ROS generation together with the lipid per-
oxidation may be the possible cause of the necrosis. In
our OGD model there is a time-dependent ROS forma-
tion during the OGD treatment and lipid peroxidation is
also induced. The ROS formation could look strange
because this treatment was performed in absence of O2
(95% N2/5 % CO2). However, mitochondrial ROS gen-
eration during hypoxia and anoxia has been shown by
several authors [24,14,40-43] although the mechanism of
this ROS generation, under this condition has not been

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 57
well resolved. During reperfusion the ROS generation, the
loss in ATP content and the lipid peroxidation reduction
are maintained below control values, which is in accor-
dance with the necrotic death that is produced during this
time, although this death is lower than during the OGD
treatment. This could mean th at, after an ischemic process,
and when the blood flow is restored, there is a group of
neurons which have suffered a big damage and that they
are unable to recover themselves, dying by necrosis.
If this reperfusion is performed in presence of 1 M
genistein, a total reversion of necrotic cell death, ROS
formation and lipid peroxidation were found as well as a
protection for increase in intracellular ATP content, in-
dicating that genistein protects against death through
reduction of both ROS and lipid peroxidation formation.
This protection against lipid peroxidation may be the
cause of a cellular membrane protection. Similar neuro-
protection against ischemia mediated by flavonoids has
been found by Zhang et al. [44] in gerbil’s brain sub-
jected to ischemia/reperfusion and by Liang et al. [24] in
brain hippocampus after global cerebral ischemia.
In the OGD model used here, apoptotic and autophagic
cell deaths were also found. Apoptotic death is probably
due to the deprivation of trophic factors during the OGD,
at least in the shortest treatments (1-5 h), because during
these times the higher caspase-3 activity is found when
cells are serum deprived and the increment due to the
OGD treatment is too low, although it is statistically sig-
nificant. However, at 24 h of OGD, additional mecha-
nisms seem to be involved since caspase-3 activity in-
duced by the OGD treatment is about a 33% higher than
the caspase-3 activity found in cells only deprived of
serum. During the reperfusion period, after 2 h of OGD,
the apoptosis is practically abolished with respect to the
cells treated with OGD. This is, perhaps, due to the
presence of trophic factors during this time. Under this
condition genistein had no additional neuroprotective
effect, result which c ould be due to the fact that apoptosis
is inhibited, under the reperfusion condition. The mecha-
nism of this apoptotic death induced by OGD, and medi-
ated by caspase-3 activation could be due to the intrinsic
mitochondrial apoptotic pathway because we found a
depolarization of the mitochondrial membrane induced
by OGD, which may show a mitochondrial membrane
alteration and, as a consequence, an influence in the cas-
pase-3 activation. Mitochondrial membrane depolariza-
tion with caspase-3 activation and cytochrome c release
was found by Figueroa et al. [25] in cortical neurons
treated with SNAP, a NO donor. However, an apoptotic
cell death mediated by extrinsic pathway could not be
discarded. Another point to comment is th e fact that dur-
ing the 24 h of reperfusion, after 1 h of OGD treatment
the mitochondrial membrane continue modified, although
in this case an hyperpolarization instead of a depolariza-
tion was observed. This mitochondrial membrane altera-
tion could also be the responsible for the necrotic death
during the reperfusion time in absence of genistein. The
fact that when reperfusion is performed in presence of
genistein there was a total reversion of necrosis could
signify that this phytoestrogen could mediate mitochon-
drial membrane reparation. This asseveration is sup-
ported by its ability to revert the mitochondrial mem-
brane potential to the control values.
The cortical neurons subjected to OGD also present a
process of autophagy because lipidation of protein LC3B
is increased. The autop hagy was more evident in the first
hour of OGD treatment, after, the autophagy decreased
but with values higher than in control cells. Under these
conditions, this autophagic process could represent a
survival mechanism induced to keep the ATP levels as it
is shown by several authors [45-47]. However, in our
case, during OGD process there is a great ATP depletion
which could indicate that in this case autophagy could
not be a protective signal. On the other hand, during
reperfusion in which cells have the enough nutrients and
oxygen, the autophagy is decreased with respect to the
OGD treatment but the values are higher than in control
cells after 1 and 3 h of OGD. On the other hand, the ATP
levels continuously decay and in this case, autophagy
only could mean a mechanism of cell death. So it would
be possible than during the reperfusion condition many
cells die not only by necrosis and apoptosis but also by
autophagia. However, when reperfusion is performed in
presence of 1 M genistein, after 1 h of OGD treatment,
a total reversion of autophagy was found, indicating that
genistein also protects against this death type, although
the molecular mechanism of this neuroprotective effect
needs to be studied. The interaction among autophagy,
apoptosis and necrosis in the hypoxia-ischemia is com-
plex and still a matter of debate and further work is nec-
essary to understand the relationships between these
three death mechanisms in brain ischemia.
In conclusion, from all these results we may infer that
the OGD treatment (O2, trophic factors, nutrients and
glucose deprivation) produces the following effects: 1)
Necrotic cell death which could be induced by the ROS
formation, lipid peroxidation increase and the ATP de-
pletion. 2) Apoptotic cell death mediated by caspase-3
activation probably through an intrinsic pathway. 3)
Autophagic cell death.
Genistein, applied during the reperfusion period, pro-
tects the cells against necrosis and autophagy, by means
of a mechanism which could be mediated by their anti-
oxidant action and by a protector effect on cellular and
mitochondrial membrane. Thus, our protective post-stress
data on neuroprotection, could serve as basis to plan a

Added after Anoxia-reoxigenation Stress, Genistein Rescues from Death the Rat Embryo Cortical Neurons 58
clinical trial with genistein in stroke patients.
5. Acknowledgements
This study was supported by the grants SAF2009-11219
and SAF2010-20337 (Ministry of Science and Tech-
nology (MCYT), Spain), CCG07-UCM/SAL-3024 (Com-
plutense University/Madrid Community) and RD06/0026/
0012 (Instituto de Salud Carlos III). E. Sánchez-Mendoza
has a contract from RD06/0026/0012. The au thors t ha n k t o
Mikel Erdocia for his help in improving the manuscript.
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