Extract
Note: Please read the complete
full text with Figures and Tables at
(in mmol/L): NaCl 119, KCl 2.5, CaCl2 2,
MgSO4 1, NaH2PO4
1.25, NaHCO3 26.2, glucose 10 (final pH 7.4). Brains were cut
coronally into 400-mm thick sections with a vibrating tissue
slicer (ZQP-86, Xiangshan, Zhejiang, China). Cortical and
hippocampal slices were quickly isolated from the
appropriate sections. Before being transferred to an experimental
chamber, all slices were incubated in ACSF bubbled with
95% O2 and 5% CO2 at 32_34 °C for 90 min recovery. After
full recovery, hippocampal slices were taken randomly into
experimental chambers perfused with ACSF. The excitation
electrode was planted in Shaffer collateral pathways of
hippocampal slices under a microscope and a recording
electrode was planted in the CA1 region. Population spikes (PS)
were recorded to monitor tissue activity before the
experi-ments. Slices were incubated with stearic acid, MK886 or
BADGE in oxygenated ACSF for 3 h, and then the activities
of the slices were evaluated by measuring the amplitude of
the PS and using the TTC staining method.
OGD injury model Brain slices were transferred to
experimental chambers and randomly assigned to 1 of 3 groups:
(i) control group, in which slices were immersed in
oxygenated ACSF at 34 °C; (ii) OGD group, in which slices were
made anoxic by re-placing the ACSF with glucose-free ACSF
equilibrated with 95% N2/5%
CO2. After 10 min insult, the slices were re-oxygenated in ACSF for 2 h; (iii) OGD+stearic
acid group, in which slices were incubated with different
concentrations of stearic acid (3_30 mmol/L) 30 min prior to
and during OGD insult.
Glutamate injury model Brain slices were transferred to
experimental chambers and randomly assigned to 1 of 3
groups: (i) control group, in which slices were immersed in
oxygenated ACSF at 34 °C; (ii) glutamate group, in which
slices were subjected to 1 mmol/L glutamate with
magnesium-free artificial cerebrospinal fluid for 30 min which had
the following composition (in mmol/L): NaCl 143, KCl 5.4,
CaCl2 1.8,
NaH2PO4 1.0,
N-2-hydroxyethylpiperazine-NĄŻ-2-ethanesulfonic acid (HEPES) 2.4, glucose 5.6, pH 7.4; (iii)
glutamate+stearic acid group, in which slices were incubated
with different concentrations of stearic acid (3_30 µmol/L)
30 min prior to and during glutamate application.
NaN3-induced injury model Brain slices were transferred
to experimental chambers and randomly assigned to 1 of 3
groups: (i) control group, in which slices were immersed in
oxygenated ACSF at 34 °C; (ii)
NaN3 group, in which slices were subjected to 10 mmol/L
NaN3 for 30 min; (iii) NaN3+stearic
acid group, in which slices were incubated with different
concentrations of stearic acid (3_30 µmol/L) 30 min prior to
and during NaN3 application.
TTC staining After insult, slices were immersed in 2%
TTC solution in a covered water bath shaker at 37 °C for 1 h
and wet weight was measured after the slices were rinsed
twice with saline. An extracting solution (50:50 mixture of
ethanol/dimethylsulfoxide) was added to the vials of slices
at a rate of 20 mL per 1 g of slice. The extracted liquid was
added to 96-well plates (200 µL per well) for 24 h and the
optical density of each well was measured at 490 nm
(OD490) by using an enzyme-linked immunosorbent assay (ELISA)
reader (Bio-TEK
Elx800uv)[13].
Blockade of the effect of stearic acid by PPAR
inhibitors MK886 is an indole compound that was originally
identified as a potent inhibitor of 5-lipoxygenase
(5-LOX)-activating protein (FLAP). Recently, it has been found to inhibit
PPAR-a via a non-competitive mechanism as shown by its
effects on the binding of arachidonic acid to
PPAR-a protein. Brain slices were treated with 5 µmol/L MK886 or 100
µmol/L BADGE (PPAR-g antagonist) in the presence or absence of
stearic acid to determine whether antagonists of PPAR could
block the neuroprotection provided by 30 µmol/L stearic acid.
The tissue activities were measured by using the TTC method.
Statistical analysis Four to six brain slices from 3 rats
were used for each experimental group, and data collected
from 6 independent experiments were used to calculate means,
which are expressed as mean±SD. SPSS statistical software
10.0 for Windows was used and statistical significance was
evaluated by using one-way ANOVA and the SNK test.
Statistical significance was assumed if P<0.05.
Results
Brain slice preparation and drug exposure The
hippocampus is very sensitive to hypoxic and chemical hypoxia.
Hypoxia and tissue injury are both associated with a
decreased amplitude of or disappearance of population spikes
(Figure 1).
Population spikes (PS) in CA1 regions of randomly
selected hippocampal slices indicated the recovery of brain
slices after incubation in ACSF for 90 min. These recovered
slices were used for the subsequent experiments. None of
the brain slices exhibited significant tissue damage
(P>0.05) after being incubated with different concentrations of stearic
acid (3_30 µmol/L), MK886 (1_10 µmol/L) or BADGE
(10_100 µmol/L) in ACSF for 3 h (as shown in Table 1). Based on
their IC50 values and our screening results, we chose
concentrations of reagents that did not produce significant
effects on tissue activity for the experiments described below.
Stearic acid at a concentration of 30 µmol/L induced a
significant increase in PS amplitude, reaching 145%±10%
(P<0.05) of basal level after 3 h treatment. Because stearic
acid may have interfered with amplitude of PS, we only
used the TTC staining method to assess tissue activity.
Neither MK886 nor BADGE affected the PS amplitude after
3 h incubation (P>0.05; Figure 2).
Brain slice insult with OGD After they were subjected
to 10 min of OGD insult and 2 h of post-incubation, the activities of the brain slices were reduced by approximately
50%. There was a statistically significant difference between
the OGD and control groups (P<0.01). Stearic acid can
dose-dependently protect against the decreases in tissue activity
induced by OGD insult (Table 2).
Brain slice insult with glutamate Only approximately
30% of tissue activity remained after brain slices were
incubated with 1 mmol/L glutamate for 30 min. There was a
statistically significant difference between the injury and
control groups (P<0.01). Stearic acid can dose-dependently
protect against the decreases in tissue activity induced by
glutamate insult (Table 2).
Brain slice insult with NaN3 After they were subjected
to 30 min exposure to NaN3, the activities of the brain slices
were reduced by approximately 50%. There was a
statistically significant difference between the
NaN3 and control groups (P<0.01). Stearic acid did not protect brain slices
from NaN3 insult (P>0.05; Table 2).
Blockade of the effects of stearic acid by PPAR
inhibitors It is known that 5-LOX inhibitor can protect against
ischemic-like injury in PC12 cells in vitro by modulating p38
MAP kinase activity[14]. Therefore, we tested whether MK886
or BADGE could directly protect brain slices against OGD or
glutamate insult. MK886 at a concentration of 5 µmol/L
significantly improved the activity in tissue insulted by OGD or
glutamate (P<0.05), whereas BADGE at a concentration of
100 µmol/L produced no significant effect on the activity of
tissue insulted by OGD or glutamate (P>0.05). When slices
were treated with 5 µmol/L MK886 during pre-incubation,
the protection against OGD or glutamate insult afforded by
stearic acid was not abolished. In contrast, 100 µmol/L
BADGE blocked its protective effect completely (Table 3).
Discussion
Experiments using brain slices have the advantages of
in vivo and in vitro studies: they not only maintain anatomic
relations and natural synaptic connectivity in
vitro, but also eliminate such in vivo variables as blood flow, temperature
and ionic environment, and closely match in
vivo conditions. Therefore, increasing numbers of brain slice models have
been used to study brain function and brain protection. In
the present study, we used 3 different damage models to
reflect the pathological characteristics of different phases of
I/R injury (metabolism disorder, toxic amino acid and
oxidative stress)[11]. The present study is the first to demonstrate
that stearic acid can dose-dependently protect rat brain slices
against OGD and glutamate toxicity, but not against
NaN3 toxicity.
As an early consequence of OGD associated with brain
ischemia, neuronal aerobic metabolism and ATP production
are severely influenced. The decrease in energy production
and malfunction of Na+/K+-ATPase leads to loss of active
ion transport, destruction of transmembrane
electrochemical ionic gradients and membrane depolarization, which
promotes presynaptic glutamate release and impaired
uptake [11]. Because brain does not rely on stearic acid oxidative
metabolism for the production of ATP, the neuroprotective
effects provided by stearic acid against OGD insult may result
from the blockage of excitatory amino acid (EAA) receptors,
defense against oxidative stress, presynaptic depression of
glutamate release or promotion of glutamate
uptake[11]. It is known that the presence of 0.5 mmol/L glutamate in the
extracellular space can reverse glutamate uptake and decrease
the synthesis of glutathione in
neurons[15]. In the present study, 1 mmol/L glutamate was applied to induce brain injury,
and stearic acid was found to dose-dependently protect brain
slices from glutamate insult. Our results suggest that the
neuroprotective effects of stearic acid are related to the
blockage of EAA receptors and/or defense against oxidative stress.
Oxidative stress is a particularly important factor in
mitochondrial dysfunction because the respiratory chain
continually leaks superoxide free radicals. These reactive
oxygen species lead to generalized oxidative damage to all
mitochondrial components. Mitochondria are the powerhouse
of the cell: their primary physiological function is to
generate ATP through oxidative phosphorylation via the electron
transport chain. NaN3, an inhibitor of cytochrome c oxidase
(complex IV of the mitochondrial electron transfer chain),
significantly decreased the tissue activity of brain slices in
the present study. In our experiments, stearic acid did not
protect brain slices against NaN3 insult, which suggests that
it acts after the malfunction of the mitochondrial electron
transfer chain during ischemia.
In some experiments, different ligands have been used
according to the different isotypes of
PPAR[16_18], and these agonists all play a role in neuroprotection to different extents.
Several fatty acids bind to all 3 PPAR isoforms, although
there is a preference of PPAR-a for polyunsaturated fatty
acids. MK886 and BADGE are 2 important antagonists of
PPAR. We found that BADGE completely blocked the
neuroprotective effect provided by stearic acid, whereas
MK886 did not abolish its protective effect. MK886 can
directly protect brain slices against OGD or glutamate insults.
Although MK886 did not abolish the effects of stearic acid,
we cannot conclude that PPAR-a is not involved in the
process of neuroprotection. However, BADGE could completely
block the neuroprotective effect of stearic acid, which suggests that PPAR-g may play a more important role in the
protective effect, because BADGE has no significant effect
on injured brain slices by itself. It has been reported that
stearic acid 200 µmol/L could only enhance reporter gene
expression under the control of ideal PPAR responsive
element (PPRE) 2-fold in HepG2 cell
clones[19]. The relationships between stearic acid and
PPAR-g or reporter gene expression under the control of ideal PPRE in neural cells have
not yet been reported on.
The findings from the present study suggest that stearic
acid can effectively protect brain during the early stages of
I/R in immature rats. Newborn animals are more tolerant than
adults of cerebral hypoxia/ischemia insult, and their
blood-brain barriers are easier to pass. Stearic acid may contribute
to protection against hypoxic-ischemic brain damage in the
immature brain.
In conclusion, stearic acid can protect brain slices (cortical
and hippocampal) against injury induced by OGD or glutamate. Its neuroprotective effect is possibly mainly
mediated by the activation of PPAR-g in brain tissue.
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