Animals and groups
We used adult male Sprague–Dawley rats which weighed between 250 and 280 g, purchased from Hebei Medical University Experimental Animal Center. All rats were kept on a 12 h-light/12 h-dark with humidity of 55 ± 5%, and had free access to food and water. Rats were randomly divided into eight groups as control (normal control), sham (sham operated), MCAO 3 h, MCAO 6 h, MCAO 12 h, MCAO 24 h, MCAO 48 h, MCAO 72 h. The brain samples were collected from the rats after decapitation. The sample size of n = 5 was estimated based power analysis taking into account our previous studies demonstrating the minimum number of samples needed to demonstrate 25% difference between treatment groups.
Permanent middle cerebral artery occlusion
Rats underwent permanent middle cerebral artery occlusion (MCAO) as previously described [4, 5]. After induction of anesthesia by intraperitoneal injection with 10% chloral hydrate (250 mg/kg), a nylon filament (diameter 0.265 mm) was inserted intraluminally into the Internal carotid artery (ICA) about 18 to 20 mm deep. Gentamicin was used to protect against infection at the incision site. Rats of the sham group received the same surgery except for the filament insertion. The body temperatures of the rats were maintained at 37 ± 1 °C by an electric blanket during the surgery. Moreover, physiological measures, including pH, pCO2, pO2, and systolic/diastolic blood pressure were recorded, and did not significantly differ among animals, confirming a homogenous set of stroke animals was enrolled in this study.
All investigators testing the animals were blinded to the treatment condition. Animals were subjected to neurological exam by using a scale for grading the ischemic injury-induced dysfunctions as follows: 0, rats extend straight both forelimbs, indicating no observable deficit; 1, rats keep the left forelimb to the breast and extend straight the right forelimb; 2, rats show decreased resistance to lateral push in addition to behavior in score 1 without circling; 3, rats twist the upper half of their body in addition to behavior in score 2. The animal was subjected to this test 3 times, and the scores from all 3 tests were averaged to give a mean neurologic deficit score (maximum possible score, 9 points divided by 3 tests = 3).
Cerebral infarction assay
The volume of infarction was analyzed using the triphenyltetrazolium chloride (TTC), a histological assay for determining dehydrogenase activity. Under deep anesthesia (chloral hydrate, 500 mg/kg, i.p.) animals were perfused intracardiacally with saline. Brains were then harvested, blocked, and processed for TTC staining. The volume of infarction was measured in each slice and summed using computerized planimetry (PC-based Image Tools software). The volume of infarction was computed as: 2 mm (thickness of the slice) × [sum of the infarction area in all brain slices (mm2)]. To minimize artifacts produced by post-ischemic edema in the infarcted area, the infarction area in the ipsilateral hemisphere was indirectly measured by subtracting the non-infarcted area in the ipsilateral hemisphere from the total intact area of the contralateral hemisphere.
The Total Protein Extraction Kit (Applygen Technologies Inc., Beijing, China) was used to obtain the total protein of the brain tissue, focusing on the peak area of ischemic tissue (bregma 1 to −1 mm). About 30 μg total protein samples were applied to electrophoresis and then transferred onto PVDF membranes (Millipore Corporation, USA) as previously described . After being blocked with 5% milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with anti-MICU1 (1:500, Santa Cruz Biotechnology) diluted in 5% milk. Polyclonal mouse anti-beta actin antibody (1:3000, Bioworld Technology) was used as a loading control. Membranes were incubated by fluorescent labeling second antibodies (IRDye 800-conjugate rabbit anti-goat IgG or rabbit anti-mouse IgG 1:5000 dilution; Rockland, Gilbertsville, PA, USA) for 1 h at room temperature. We analyzed the relative density of bands on the Odyssey infrared scanner (LICOR Bioscience, Lincoln, NE, USA).
Paraffin-embedded sections were used to evaluate the expression of MICU1 at 6 h and 12 h after MCAO as previously described . The brains of the rats were fixed in 4% paraformaldehyde over 24 h at 4 °C, embedded in paraffin after being dehydrated in different gradients of alcohols. Then cut the brain into 5 μm per slice. Brain sections were blocked in 3% normal donkey serum and then incubated with MICU1 goat polyclonal antibody (1: 100, Santa Cruz Biotechnology) overnight at 4 °C. The secondary antibodies were used the next day (Zhongshan Biology Technology Company, Beijing, China). Five visual fields of the ischemic regions (bregma 1 to −1 mm which corresponded to core and peri-infarct areas to fully capture the extent of cell death) were selected to count the mean density value under a 400× microscope. A separate cohort of animals were also subjected to MCAO, with animals euthanized at 6 h and 12 h after stroke, and brains harvested as above, but processed for double-labeling of MICU1 (1: 100, Santa Cruz Biotechnology) and the neuronal marker MAP2 (1:100 Abcam). The regions of interest for estimation of MICU1 expression and MAP2 positive cells included the peak area of ischemic regions (bregma 1 to −1 mm), which were photographically captured (Axiophot2; Zeiss), and cells were quantified by counting per HPF view selected at random and corrected by the Abercrombie formula. Six captured fields in each coronal level, using three anterior-posterior levels, were used to analyze the MICU1 expression and MAP2 positive cells, using Scion Image software (Scion, Frederick, MD). Binary images were created using a distinct threshold, and then the positive areas were calculated.
Real-time quantitative polymerase chain reaction
Rats’ brain tissue, also focusing on the peak area of ischemic tissue (bregma 1 to −1 mm), was rapidly dissected and the total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA). RNA (2 μg) from each sample was applied to reverse transcribed for synthesized cDNA. Primers were synthesized by Shanghai Biological Engineering Technology Company Limited. Forward and reverse primers were 5’-TTCCTCACAACGGTGCTCTC-3’ and 5’-CCAGACTTGAGGGTGTTCCC-3’ for MICU1 and 5’-CCCATCTATGAGGGTTACGC-3’ and 5’-TTTAATGTCACGCACGATTTC-3’ for β-actin. Each sample was measured in triplicate and all samples were normalized by β-actin. Relative gene expression was calculated with the 2-ΔΔCT method and then analyzed using logarithmic transformation.
Data and statistical analysis
All results are shown as mean ± standard error. One-way ANOVA followed by Student-Newman-Keuls and LSD tests were applied for multiple comparisons. Mann–Whitney U test was applied for neurological deficit score. The differences with P < 0.05 were considered statistically significant.