Animals and treatment
C57BL/6J mice weighing 18-22g were provided by the Reference Animal Research Centre of Yangzhou University (Yangzhou, People’s Republic of China; certificate no SCXK 2014-0004). All procedures and assessments were approved by the Animal Ethics Committee of the School of Chinese Materia Medica, China Pharmaceutical University. These experiments were carried out in accordance with the National Institutes of Health Guide for the Care and use of Laboratory Animals (National Institutes of Health Publication No 80-23, revised in 1996). Before performing the experiments, all animals were randomized into experimental groups, and the indices were measured by operators blinded to the study procedures.
Middle cerebral artery occlusion(MCAO) model
A middle cerebral artery occlusion (MCAO) model was induced using the modified intraluminal filament method as follows: for blocking the blood supply to the ipsilateral hemisphere of the rat brain, the right middle cerebral artery was occluded by inserting a 4-0 nylon monofilament suture into the right internal carotid artery. The body temperature was sustained at 37℃ with a heated blanket throughout the procedure. The animals underwent 1h of MCAO and then were reperfused by careful withdrawal of the filament. In the sham group, an identical surgical procedure was performed without disturbing the arteries.
Oxygen and glucose deprivation/reperfusion(OGD/R)model
In order to generate I/R-like conditions by OGD/R in vitro, bEnd.3 cell was placed in a 37°C anaerobic chamber (0.2% O2, 5% CO2, 95% N2) and cultured in glucose-free medium for 6h. After the oxygen-glucose deprivation, the cells were placed in glucose-containing DMEM with 10% FBS and then incubated under normoxic conditions for 24 h in order to affffect reperfusion. Control samples were taken from cells cultured under normal conditions.
Rus was labeled with Tc99m using tricine and EDDA as the coligands. The radiolabeling procedure was the literature method. In brief, to a clean vial were added 30 μL of a Rus solution (1 mg/mL in 30% ethanol), 200 μL of a tricine solution [100 mg/mL in 0.1 M phosphate buffffer (pH 6.0)], 25 μL of a SnCl2 solution (1.0 mg/mL in 0.1 N HCl), and 100 μL of a Na2Tc99mO4 solution (370 MBq) . The reaction mixture was kept at room temperature for 10 min. To the solution were added 500 μL of EDDA [20 mg/mL in 0.1 N HCl (pH 1.0)] and 250 μL of Na2HPO4 [0.2 M in H2O (pH 8.5)]. The vial containing the reaction mixture was sealed, cramped, and heated at 60℃ for 40 min. After being cooled to room temperature, a sample of the resulting solution was analyzed by radio-HPLC with HPLC method. The structure is shown in Figure 1. The synthesis of Rus-Tc99m and its radiolabeling were achieved as mentioned.
Brains were quickly removed at 12 h post-ischemia. The 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining was performed to evaluate tissue viability and measure the infarct size. The infarct area was measured in NIH Image J software (Version 1.42; National Institutes of Health, Bethesda, Md). The infarct areas on each slice were summed and multiplied by slice thickness to give the infarct volume. Infarct volume was expressed as a percentage of infarction per ipsilateral hemisphere.
Cells were incubated in the Millicell bEnd.3 cells culture inserts in a humidified atmosphere of 5% CO2 and 95% air for 7d. After OGD/R condition, the medium was removed. 100 μL of EB & Rus-Tc99m solution were added into the Millicell cell culture inserts and 1 mL of Krebs buffer were added into the external chamber. The cells were continuously incubated for another 60 min and then the external Krebs solution were collected and added to 96-well black microplate. EB concentration value was measured at an excitation wavelength of 620 nm, with an emission wavelength of 530 nm using an Infinite M200 Pro plate reader (Tecan, NC, USA), radioactive counting value was measured by γ-ray counter.
Evans Blue (EB) in vivo
BBB permeability was assessed by the leakage of EB stain into the brain following the tail-vein injection. Two hours before the animals (n=6 for each group) were euthanized, 0.1 mL per 10 g body weight of 2% EB (Sigma Aldrich) in normal saline was injected into each animal. The animals were then anesthetized and perfused with normal saline. For the quantitative measurement of EB leakage, the ipsilateral hemisphere was removed and homogenized in 1 mL of trichloroacetic acid, then centrifuged at 12,000 g for 20 min. EB concentration was quantitatively determined by measuring the absorbance at 620 nm of the supernatant with a spectrophotometer. The EB content was quantified as micrograms of EB per gram of tissue, using a standard curve.
Cerebral blood flow (CBF) was measured using laser Doppler flowmetry. A computer-controlled optical scanner directed a low-power laser beam over the exposed cortex. The scanner head was positioned parallel to the cerebral cortex at a distance of ~20 cm. A color-coded image indicating specific relative perfusion levels was displayed on a video monitor. The images were acquired at the onset of ischemia and reperfusion 0, 6, 12 and 24 h (n=6 for each group).
Neurological deficits of the experimental animals were graded on an 18-point scale as previously described. The measurement of neurological deficits consisted of the following tests: spontaneous activity, symmetry of movements, symmetry of forelimbs, climbing, reaction to touch, and response to vibrissae touch. All six individual tests were scored on a four-point scale as 3, 2, 1, or 0. Final score was obtained by adding the scores recorded for each individual test, with a maximum score of 18 observed in healthy animals.
In vivo SPECT/CT
All in vivo imaging experiments were conducted on a dedicated small animal SPECT system (NanoSPECT/CT, Mediso) in Xiamen university, center for molecular imaging and translational medicine, calibrated for technetium-99m (Tc99m) emitted γ-radiation. Tracers were applied in amounts, corresponding to 10 MBq/animal intravenously into the tail vein 24 h before in vivo imaging. The mean scan time was about 60 min with an initial morphologic whole body spiral CT and a consecutive whole body SPECT scan with a frame time of 60 s. Animals were held under Isoflurane inhalation anaesthesia (2% Isoflurane in air) for the whole scan time. Data were reconstructed and analysed using in built VivoQuant Software and presented as percentage of the injected dose (%ID) per selected region of interest (ROI). For individual organ analysis, 3D ROIs were drawn on the anatomic CT images. The ROI size was identical for all parallel experiments. Mean values and standard deviation were calculated for %ID. After in vivo imaging, mice were either kept for longitudinal follow up examinations or sacrificed for tissue collection.
Western blot analysis
The cells or samples (n = 6, for each group) were decapitated and rapidly collected. The prepared cells or tissues were homogenized in 1:10 (w/v) ice-cold protein extraction buffer in glass homogenizers. Soluble proteins were collected and centrifuged at 12,000 g for 10 min at 4℃, and then the supernatant was used to detect the level of ZO-1, occludin and the total protein. The protein concentration of the samples was determined by BCA protein assay reagent kit. Equal amount of protein lysate (50 μg) in each group was separated by 8% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins on the gel were subsequently transferred onto a nitrocellulose membrane (260 mA, 2 h). The membranes were blocked with PBST containing 5% skim milk for 2 h at room temperature and then incubated with primary rabbit monoclonal antibody respectively overnight at 4℃(ZO-1, occludin, 1:800; Proteintech Group, USA). The membranes were then washed and incubated with secondary antibody (anti-rabbit IgG, 1:3000; Proteintech Group, USA) for 1.5 h at room temperature. The anti-actin antibody (1:1000; Proteintech Group, USA) served as control. The protein bands were visualized with enhanced chemiluminescence reagents (ECL), and the signal densitometry was quantified by an observer blinded to the groups of the animals using western blotting detection system (Quantity One, Bio-Rad Laboratories, USA).
All results are expressed as mean±standard deviation (SD). GraphPad Prism (GraphPad software, San Diego, CA, USA) was used for statistical analysis of the data. The signiﬁcant of the differences between two groups was analysed using unpaired Student’s t test, and multiple comparisons was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. All tests were two-tailed. P<0.05 was considered statistically signiﬁcant.