Male C57BL/6 mouse were anesthetized by intraperitoneal injection of chloral hydrate (353 mg/kg) with using the doppler excitation photo flowmeter monitors cerebral blood flow in cerebral cortex supplied by middle cerebral artery (MCA). The head was fixed on the animal skull, at the position of 2 mm after the anterior halogen, 6 mm to the right of the midline. Short focal cerebral ischemia, cerebral artery occlusion (MCAO) induction. The specific procedure was as follows: the common carotid artery was exposed and separated on the supine position of mice, the distal end was ligated, the proximal end was clamped with hemostatic forceps, a small incision was made with microsurgical scissors, and a 6-0 nylon monofilament suture was inserted into the internal cervical movement pulse 10 mm to block MCA source. After 60 min of ischemia, the single thread was removed to complete the reperfusion. The body temperature of the animals was maintained at 37 oC by a heating lamp (FHC, Bowdoinham) at the middle and 2 h after the start of reperfusion. At the beginning of reperfusion, 7 was injected into the lateral ventricle of mouse. In order to determine the volume of cerebral infarction, mouse was anesthetized 24 h after surgery, and their heads were severed TTC staining was performed on coronal sections. The calculation formula of infarct volume percentage was: (The unstemmed areas on the left - right unstemmed area)/left unstemmed area*100%. The experiment was divided into three groups: control (sham operation), model group, medium-low temperature protection group .
In the present study, neurological function score and cerebral infarction volume measurement were used to detect ischemia reperfusion injury in mouse. The expressions of oxidative stress related factors (ROS, MDA, SOD, GSH-px) in mouse were detected. Western blot was used to detect the expression of autophagy marker lc3-II/I in mouse of the three groups, and to observe and detect the autophagy status of mitochondria in brain tissues by electron microscopy. Mouse were treated with SIRT1 inhibitor (Sirtinol) and divided into control group (sham operation), model group, medium-low temperature protection group, medium-low temperature protection + Sirtinol. WB and PCR expression of SIRT1, FOXO1 and PINK1/Parkin related proteins, autophagy lc3-II /I proteins, oxidative stress and cerebral infarction volume in the brain tissues of mice in each group.
A hypoxia/reoxygenation model was established using mouse hippocampal neuron cell HT22 cell line to simulate ischemia reperfusion injury. Group 1) control; 2) model; 3) model +SIRT1; 4) model + SIRT1 + FOXO1. Transfection of SIRT1 and FOXO1 and expression of SIRT1, FOXO1 and PINK1/Parkin related proteins were detected in each group. The expressions of oxidative stress related factors and autophagy lc3-II/I protein in each group were detected.
In this study, TTC staining was used to measure the volume of cerebral infarction. 4% chloral hydrate anesthesia (100 mg/kg), quickly remove brain, remove the olfactory bulb and hindbrain, taking four starting frontopolar coronary slices, about 1.5 mm thick, immediately put 2% TTC solution (0.01 M phosphate buffer preparation pH7.4 slightly), 37 ℃ avoid light incubation for 30 min. Then soak and fix with 10% paraformaldehyde solution. Infarcted areas appear white and non-infarcted areas appear red. Champion Image hpias-1000 high-resolution color pathological Image and text report analysis system was applied to measure the area of cerebral infarction in each area, and the infarct volume was calculated according to the formula V= t (A1+… An) - (A1+ An) t/2.Where t is the slice thickness and A is the infarct area .
Expression detection of oxidative stress related factors
Levels of oxidative stress related factors were measured using ELISA kit (Cyman system) according to the instruction manual.
Samples were prepared by lysis with proteinase inhibitor. The protein was quantified by BCA kit (Pierce Biotechnology, IL). Each sample were separated by 15% SDS-PAGE, and the membranes were incubated for 12 h at 4°C with antibodies, and then incubated with goat-anti-mouse IgG (Santa Cruz Biotechnology USA). The signals were detected with ECL reagents.
Real-time reverse transcription-polymerase chain reaction
Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The concentration of RNA was measured using a NanoDrop-1000 (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was determined by 1.5% agarose gel electrophoresis. cDNA synthesis was performed according to the manufacturer's recommendation. Briefly, RNA was reverse-transcribed (RT) using a ReverAid First Strand cDNA kit (Thermo Fisher Scientific). Under our experimental conditions, we used β-actin as an internal control for normalizing the expression levels of mRNA. cDNA products were then diluted at 20X, and the 10-μl PCR mixture contained 1 μl diluted RT product, 5 μl SYBR-Green Master Mix, 2 μl RNase-free water, 1 μl forward, and 1 μl reverse primers. The reaction was incubated at 95˚C for 10 min followed by 40 cycles of 95˚C for 15 sec and 65˚C for 60 sec. The relative mRNAs expression was calculated using the 2-ΔΔCt method.
Electron microscopy observation
Placed the paraffin tissue section in room temperature 20 min, the tissue sections were soaked in xylene for 10 min, and then soaked for 10 min after replacing xylene, soak in anhydrous ethanol for 5 min, immerse in 95% ethanol for 5 min, soak in 70% ethanol for 5 min. Boiling hot fix, electric or water bath pot heating 0.01 M sodium citrate buffer (pH6.0), in a tissue section heating 10-15 min. Transparent at room temperature for 15 min in transparent solution. The slices were placed in the sealing solution, sealed at room temperature and away from light for 30 min, and the excess liquid was thrown away. Observed the results under the electron microscopy and take photos.
mimics and negative control oligonucleotides were synthesized by GenePharma Co., Ltd. (Shanghai, China). Cell transfection was performed using Lipofectamine 2000 reagents (Invitrogen Life Technologies) according to the manufacturer's protocol. Twenty-four hours after transfection, cells were collected for RT-PCR, Western blot analysis and functional assay.
Results were performed using SPSS 19.0, and presented as the mean ± SD from three independent experiments. A value of P < 0.05 was considered to indicate a statistically significant difference.