Supplementary Materials and Methods
Infarct size assessment
On post-operative day (POD) 3, animals were decapitated and 2-mm thick coronal sections
from throughout the brain were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC,
Amresco Inc., USA) to evaluate the infarct volume. To compensate for the effect of brain edema,
the corrected infarct volume was calculated as previously described: corrected infarct area =
left hemisphere area − (right hemisphere area − infarct area) (1). Infarct volume was manually
quantified using ImageJ software and expressed as a percentage of the contralateral structure.
Behavioral tests
A battery of behavioral tests including modified neurological severity scores (mNSS) (2) and
modified sticky-tape test (MST) (3) were performed before MCAO and at PODs 1, 3, 7, 14, 21,
28 and 42 by two investigators (Q.Y. and X.K.) who were blinded to the experimental groups. All
behavioral tests were conducted in the light phase at approximately the same time each day.
mNSS
mNSS is a composite of motor, sensory, reflex, and balance tests. Neurological function
was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18). In the severity
scores of injury, 1 score point is awarded for the inability to perform the test or for the lack of a
tested reflex; thus, the higher score, the more severe is the injury.
MST
1
To assess the somatosensory dysfunction following cerebral ischemia, we performed the
modified adhesive removal (sticky-tape) test. Briefly, a non-removable tape sleeve is wrapped
around the animal’s paw as a tactile stimulus. When placed in a testing cage, the rats will use
their mouth, and to a lesser extent the other forelimb, to attempt to remove the labels. The time
spent attempting to remove the stimulus was recorded during a 30-s observation period. The
test was repeated on the opposite limb until sessions consist of five trials per limb each day for
a total of ten calculated ratios. The average ratio of left to right performance was calculated as
the ratio of interest.
Isolation of mitochondria
Mitochondria were isolated according to the procedure described by Clark and Nicklas (4).
Rats were decapitated and the brains were immediately removed and homogenized in an
ice-cold isolation buffer (0.25 M sucrose, 1 mM K-EDTA, 10 mM Tris-HCl, pH 7.4). The
homogenate was immediately centrifuged at 2000 g for 3 min, the supernatant was centrifuged
again at 2000 g for 3 min, and the second supernatant was decanted and centrifuged at 12,000
g for 8 min. The supernatant was discarded and the pellet was resuspended in isolation buffer
without K-EDTA. Then the suspension was centrifuged at 12,000 g for 10 min. The resulting
brown mitochondrial pellet was resuspended in the same buffer. All the above procedures were
carried out at 0–4 °C.
Determination of mitochondrial membrane potential (MMP) and respiratory chain
complexes activities
2
The ischemic core and penumbra in the ipsilateral hemisphere to the occlusion, and
contralateral brain tissue were dissected at 4 h and 24 h after reperfusion as described (5-6). All
the enzymatic activities were measured at 37 °C spectrophotometrically (7) by the following
methods: complex I (NADH dehydrogenase) (8), complex II (succinate dehydrogenase) (9-10),
complex III (ubiquinol cytochrome c reductase) (11), and complex IV (cytochrome c oxidase)
(12). MMP was determined using JC-1 (Sigma, USA). Mitochondria samples (0.5 mg/ml, 1 ml)
were incubated with 19 ml JC-1 staining buffer according to the manufacture’s instruction
(Sigma, USA). At the end of the experiments, valinomycin was added as a negative control.
Fluorescence intensity was determined at 37°C in a fluorescence spectrophotometer (Tecan,
Switzerland). The ratio of aggregates (red, 590 nm) to monomer (green, 525 nm) was
calculated as an indicator of MMP.
Mitochondrial ROS production
Mitochondrial ROS production was measured using the indicator dichlorodihydrofluorescein
diacetate (H2DCFDA; Invitrogen, USA) as we described earlier (13).
Opening of mPTP in isolated mitochondria
Opening of mPTP was determined by Ca2+-induced mitochondrial swelling (14). The
decrease in light scattering closely parallels the percentage of the mitochondrial population
undergoing permeability transition. Briefly, 1 mg of isolated mitochondria was suspended in 1
ml of respiration buffer plus 10 mM succinate. After a 5-min pre-incubation at 36°C and baseline
3
measurement, CaCl2 was added to the cuvet. Mitochondrial swelling was measured as a loss of
absorbance (540 nm) using a spectrometer following the addition of Ca 2+.
Western blot analysis
Penumbra brain samples (5) were fractionated into the cytosolic, mitochondrial, and nuclear
fractions using the Pierce Mitochondrial Isolation Kit and NE-PER Nuclear and Cytoplasmic
Extraction Kit (Thermo, USA) according to the manufacturer’s protocols. Western blot was
performed as described before (15). We used primary antibodies to cytochrome c (CytoC;
Santa Cruz, USA, 1:400), apoptosis-inducing factor (AIF; Cell Signaling, USA, 1:1000), cleaved
caspase-3 (Cell Signaling, USA, 1:1000), caspase-3 (Cell Signaling, USA, 1:1000), cleaved
caspase-9 (Cell Signaling, USA, 1:1000), caspase-9 (Cell Signaling, USA, 1:1000), COX IV
(Abcam, UK, 1:1000), and Histone H3 (Abcam, UK, 1:2000).and β-actin (Santa Cruz, USA,
1:3000). Secondary antibodies were all purchased from GE Healthcare (Buckinghamshire, UK):
peroxidase labeled anti-mouse antibody (1:5000), anti-rabbit antibody (1:10000), anti-goat
antibody (1:5000). Specific signals of proteins were visualized by chemiluminescence using the
ECL western blotting detection system (GE Healthcare UK). Densitometry of Western blots was
analyzed with Quantity One software and normalized to respective loading control signal on
each blot.
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