In a temperature control room (23-25°C), adult male Sprague-Dawley (SD) rats were housed in three rats per cage at a 12 h light/dark cycle, weighing 230 to 250 g, free access to drinking water and food. Adapt to the environment at least 3 days before the experiment. All animal use and experimental protocols were approved and carried out in compliance with the IACUC guidelines and the Animal Care and Ethics Committee of Wuhan University School of Medicine and Qingdao University School of Medicine. Randomization was used to assign samples to the experimental groups, and to collect and process data. The experiments were performed by investigators blinded to the groups for which each animal was assigned.
Local cerebral ischemia and infarct size measurement
Transient cerebral ischemia induced by suture occlusion technique . Male SD rats in each group were randomly sampled to select rats with the same weight range for experiment. Male SD rats were anesthetized with a mask of 4% isoflurane in 70% N2O and 30% O2. The midline neck incision, careful exposure and dissection of the right external carotid artery (ECA), from the ECA insertion line into the right internal carotid artery occlusion of the right middle cerebral artery (MCA) (about 22 mm). After occlusion for 90 minutes, the wire plug was removed to allow reperfusion, the ECA was ligated, and the wound was closed. Sham-operated rats underwent the same surgery and/or intraventricular injection, except that the plug was inserted and immediately withdrawn. The body temperature was maintained at 37.0 ± 0.5°C using a heating pad and a heat lamp. 24 hours after MCAO, after anesthesia with 4% isoflurane in 70% N2O and 30% O2, rats were reperfused with ice-cold 0.9% saline, and then the brain was quickly removed for Western blotting and TTC (2,3,5-triphenyltetrazolium chloride) staining. The rats were sacrificed (n = 56) and the brains were removed for Western blotting. Tissues surrounding the infarcts of the ipsilateral hemispheres were homogenized in RIPA buffer for 30 minutes on ice using a tissue mill. Tissue lysates were then centrifuged at 12000×g for 15 minutes at 4°C and all protein concentrations were determined using a BCA protein assay device. The rats were sacrificed (n = 40) and the brain was removed for TTC staining to assess the volume of cerebral infarction . The brain was placed in a cooled matrix and cut into 2 mm coronal sections. Each section was placed in a 10 cm dish and incubated with 2% TTC in phosphate buffered saline for 30 minutes in an oven at 37 °C. The sections were fixed in 4% paraformaldehyde in a 4°C refrigerator. All image acquisition, processing and analysis in a blind manner and under controlled environmental lighting. Scanned images were analyzed using ImageJ software and infarct data from all groups were expressed as the ratio of infarcted area to total brain slice area .
Intraventricular injection (i.c.v)
The rats were anesthetized with a mixture of 30% O2 and 70% N2O with 4% isoflurane in a sealed fluoroscopy box. When the rats are deeply anesthetized, we will use the ear bars and the upper incisor bar to fix the rat's head in the stereotactic frame，Rats were anesthetized continuously with a 4% isoflurane mask. Next, a small sagittal incision was made in the rat's head and the anterior iliac crest was positioned as an anatomical reference point. The ventricles (from the anterior iliac crest: 1.5 mm; anteroposterior, - 0.8 mm; depth, 3.5 mm) were connected to a Hamilton microinjector via a polyethylene tube using a 23-gauge needle at a drug infusion rate of 8 μl/min for 2 min. Appropriate needle placement was verified by taking a few microliters of clear cerebrospinal fluid into a Hamilton microsyringe.
Cortical Neuron Culture and OGD Insult
Cortical neuronal cultures were prepared from SD rats on day 17 of gestation as we described in previous reports. Pregnant rats were anesthetized with 4% isoflurane in 70% N2O and 30% O2 and sacrificed by cervical dislocation. The rats were spray-sterilized with 70% ethanol and the embryos were removed. Quickly break the embryo and place the cortex in the cold after removing the meninges plating medium（neurobasal medium, 2% B-27 supplement, 0.5% FBS, 0.5 mM L-glutamax and 25 mM glutamic acid）. Cortical neurons were suspended in a plating medium and plated on a Petri dish coated with poly-D-lysine. Half of the plating medium was taken out and replaced with maintenance medium (Neurobasal medium, 2% B-27 supplement, and 0.5 mM L-glutamine) in the same manner every 3 days. After 12 days, the cultured neurons were used for experiments . For OGD/R injury, the cells were transferred to a deoxygenated, glucose-free extracellular solution (in mmol/L: 116 NaCl, 5.4 KCl, 0.8 MgSO4, 1.0 NaH2PO4, 1.8 CaCl2, and 26 NaHCO3); into a specialized, humidified chamber and maintained at 37 °C, 85% N2/10% H2/5% CO2 for 60 minutes. The medium was then replaced with fresh maintenance medium containing the appropriate concentration of reagents in a 95% O2/5% CO2 incubator for 24 hours during recovery. First transfer the control culture to another extracellular solution（in mmol/L: 116 NaCl, 5.4 KCl, 0.8 MgSO4, 1.0 NaH2PO4, 1.8 CaCl2, 26 NaHCO3, and 33 glucose）, and then introduced into the humidified chamber which were maintained at 37°C for 60 minutes in 95% O2/5% CO2. Then the medium was replaced with fresh maintenance medium for the whole period at 37°C in a 95% O2/5% CO2 incubator.
tDCS and DCS experimental procedures
For mice MCAO model, tDCS was applied in mice without re-anesthesia by a constant current stimulator (Schneider Electronics, Gleichen, Germany) that was specifically designed for application of low-intensity currents in small mammals . Epicranial electrode implant was carried out in mice 7 days before the MCAO operation. One electrode was positioned on each side of the cranium in a symmetrical way and fixed with nontoxic glass ionomer cement. The electrode over the ischemic cortex was connected to the cathodal terminal and the other electrode was connected to anodal terminal. Prior to stimulation, epicranial implanted electrode was filled with saline solution. The contact area of the electrode toward the skull was 3.5 mm2. The tDCS was applied in mice at current intensity of 100 μA with a current density of 2.86 mA/cm2. Mice underwent tDCS at 3 h after I/R for 10 min, followed by 3 min rest and then 10 min stimulation, for a total 8 times of 10 min stimulation. To avoid a stimulation break effect, the current strength was ramped for 10 s. The sham mice underwent the same procedure of stimulated groups, but no current was applied.
For OGD model, steady DCSs at the physiological strength 250 mV/mm were applied to cultured neurons in culture chambers using methods described previously . For the DCS stimulation, agar-salt bridges were used to connect silver/silver chloride electrodes in beakers of Steinberg’s solution, to pools of excess culture medium at either side of the chamber. Field strengths were measured directly at the beginning and end of the observation period. Culture conditions in control were identical except no DCSs were applied. HEPES acid (20 mM) was added to the culture medium, with pH adjusted to 7.4. The cells were stimulated by DCS at the current strength of 250 mV/mm for 20 min at 3 h after reoxygenation following OGD.
Western blotting analysis as described previously . In short, the polyvinylidene difluoride membrane (Millipore, USA) was incubated with primary antibody (Cezanne mouse monoclone antibody, 1:1,000, cat. no. sc-514402; Santa Cruz Biotechnology; SIRT6 rabbit monoclonal antibody, 1:1,000, cat. no. ab191385; Abcam; p53 rabbit polyclonal antibody, 1:1,000, cat. no. #9282; Cell Signaling Technology; caspase-3 mouse monoclonal antibody, 1:1,000, cat. no. 31A1067; Novus Biologicals; Ku70 and Ku86 mouse monoclone antibody, 1:1,000, cat. no. sc-17789 and sc-515736; Santa Cruz Biotechnology；GAPDH mouse monoclonal antibody, 1: 3,000, cat. no. sc-365062; Santa Cruz Biotechnology) overnight at 4°C. Primary antibodies were labeled with horseradish peroxidase-conjugated secondary antibody, and protein bands were imaged using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, USA). The EC3 Imaging System (UVP, USA) was used to obtained blot images directly from the polyvinylidene difluoride membrane. The experimenters were blinded to the groups allocation during the experiment. The quantification of Western blot data was performed using ImageJ software.
Rats were treated with an over dose of isoflurane, then intracardiac perfusion with 0.9% saline, next put in 4% paraformaldehyde (PFA) at 4°C for 24 h, and followed by transfered into 30% sucrose solution in 100 mol/mL phosphate buffer at 4°C for 72h. Then the brains tissue of rats and human were kept in 4% paraformaldehyde solution at 4°C overnight. Brains tissues were cut into 16 µm coronal sections by a Leica VT1000S vibratome (Leica Micro-systems AG, Nussloch, Germany). The brain sections were treated with primary antibody rabbit anti- Cezanne (1:100) from 1:100, Wuhan sanying Biotechnology, China, mouse anti- SIRT6 (1:100) from 1:100, Santa Cruz Biotechnology, USA, rabbit anti- NeuN (neuronal-specific nuclear protein) and mouse anti- NeuN from 1:100, Abcam, USA. The secondary antibody goat anti- Rabbit 488, anti- Mouse 488, goat anti- Rabbit 594,goat anti- Mouse 594 from Molecular Probes (Eugene, USA). The sections were photographed by a blinded investigator using an Olympus fluorescent microscope (IX51, Olympus, Japan). Analysed by Image J software (Image J, USA).
Analysis of lactate dehydrogenase release and cell viability
Lactate dehydrogenase (LDH) release was analyzed using a colorimetric CytoTox 96 Cytotoxicity kit (Promega). Cell viability in the neuronal cultures was evaluated by the ability to take up thiazolyl blue tetrazolium bromide (mingest thiazolyl tetrazolium, MTT) (PowerWave X; BioTek, Winooski, VT). The two methods were performed following the manufacturer’s instructions.
shRNA(sh)-Cezanne Lentiviral Particles (cat.no.sc-151945-V; Santa Cruz Biotechnology, Inc.) or Control shRNA Lentiviral Particles (cat.no. sc-108080; Santa Cruz Biotechnology, Inc.) were transfected in primary neurons according to the manufacturer’s protocol. Prior to transfection, cells were plated in 6-well or 96-well plates and grown to 40-50% confluence. Cells treated with 10 μl/ml sh-Cezanne or Control shRNA Lentiviral Particles were transfected for 96 h. Select stable clones expressing the shRNA-Cezanne via Puromycin dihydrochloride (cat.no.sc-108071; Santa Cruz Biotechnology, Inc.) selection.
shRNA(sh)-Cezanne Lentiviral Particles (cat.no.sc-151945-V; Santa Cruz Biotechnology, Inc.) or Control shRNA Lentiviral Particles (cat.no. sc-108080; Santa Cruz Biotechnology, Inc.) were transfected according to the manufacturer’s protocol.The rats received 16 µl lentiviral supernatant (109 infectious units/ml) by intracerebroventricular (i.c.v) injection.
shRNA(sh)-SIRT6 Lentiviral Particles (cat.no.sc-63029-V; Santa Cruz Biotechnology, Inc.) or Control shRNA Lentiviral Particles (cat.no. sc-108080; Santa Cruz Biotechnology, Inc.) were transfected in primary neurons according to the manufacturer’s protocol. Prior to transfection, cells were plated in 6-well or 96-well plates and grown to 40-50% confluence. Cells treated with 10 μl/ml sh-SIRT6 or Control shRNA Lentiviral Particles were transfected for 96 h. Select stable clones expressing the shRNA-SIRT6 via Puromycin dihydrochloride (cat.no.sc-108071; Santa Cruz Biotechnology, Inc.) selection.
The transfection of the SIRT6 plasmid (Addgene) was conducted using the X-tremeGENE HP DNA Transfection Reagent (Roche) in terms of the manufacturer’s instructions. A density of 2×105 cells was first seeded in each well of a six-well plate and then transfected with complexes containing 2 μg of SIRT6 plasmid or a negative control with pcDNA3.1 and 2 μl of the X-tremeGENE transfection reagent. Then, the cells treated with1μg/μl pcDNA3.1-SIRT6 or negative controlwere incubated under normal condition for 48 h at 37°C.
Total 62 rats were used for neurobehavioral tests. Neurological Severity Scores: The rats were subjected to a modified neurological severity score (mNSS) test as reported previously . These tests are a battery of motor, sensory, reflex, and balance tests, which are similar to the contralateral neglect tests in humans. Neurological function was graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18).
Beam walk test: The beam walk test measures the animals’ complex neuromotor function . The animal was timed as it walked a (100 x 2 cm) beam. A box for the animal to feel safe was placed at one end of the beam. A loud noise was created to stimulate the animal to walk toward and into the box . Scoring was based upon the time it took the rat to go into the box. The higher the score, the more severe is the neurological deficit.
Adhesive-removal test: A modified sticky-tape (MST) test was performed to evaluate forelimb function . A sleeve was created using a 3.0 × 1.0cm piece of yellow paper tape and was subsequently wrapped around the forepaw so that the tape attached to itself and allowed the digits to protrude slightly from the sleeve. The typical response is for the rat to vigorously attempt to remove the sleeve by either pulling at the tape with its mouth or brushing the tape with its contralateral paw. The rat was placed in its cage and observed for 30s. Two timers were started: the first ran without interruption and the second was turned on only while the animal attempted to remove the tape sleeve. The ratio of the left (affected)/right (unaffected) forelimb performance was recorded. The contralateral and ipsilateral limbs were tested separately. The test was repeated three times per test day, and the best two scores of the day were averaged. The lower the ratio, the more severe is the neurological deficit.
Student’s t test or ANOVA test was used where appropriate to examine the statistical significance of the differences between groups of data. All results are presented as mean ± SEM. Significance was placed at p < 0.05.