Animals
Adult male Sprague-Dawley (SD) rats (280 ± 20 g) housed under controlled conditions with a 12-h light/dark cycle, a temperature of 21 ± 2°C, and humidity of 60–70% for at least 1 week before drug treatment or surgery, purchased from the Experimental Animal Center of the Fourth Military Medical University (Xi’an, Shaanxi, China) and the Laboratory Animal Center of Xi’an Jiaotong University (Xi’an, Shaanxi, China). It was approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University and by the Institutional Animal Care and Use Committee at Xi’an Jiaotong University. Every effort was made to minimize stress to the animals. All sample sizes for the assessment parameters were calculated to reduce the number of animals used.
Transient Focal Cerebral Ischemia Mode
Focal cerebral ischemia was induced in the rats by middle cerebral artery occlusion (MCAO), as described previously[12, 13]. The experimental animals were randomly divided into 3 groups, with 10 animals in each group. Because the success rate of the MCAO model is about 70%, the actual number of animal samples used in each group is 8 ~ 6 (n = 8 ~ 6 for each group). The three groups were divided into the Sham group, the MCAO group, and the MCAO + TAT-PEP group. In the MCAO + TAT-PEP group, 1.0 mg/kg TAT-PEP was intraperitoneally injected immediately after reperfusion, and then TAT-PEP of this concentration was injected daily.
Garcia scores
At 24h, 48h, and 72h after MCAO, the modified Garcia scores [14] were used to detect the neurological function of experimental animals. It contains 18 points, including 0–3 points for each item, as shown below. Free activity in a cage for 5 min: no activity (score 0); Almost unable to move (score 1 point); Able to move, but the range of movement does not reach 3 sides in the cage (2 points); The field of activity shall get at least 3 sides inside the enclosure (3 points). The symmetry of limb movement: no movement of the left limb (0 points); The left limb can move slightly (1 point); The left limb can move slowly (2 points); Bilateral limb movements are symmetrical (3 points). The symmetry of forelimb (forelimb extension when lifting the tail): the left limb is unable to move (0 points); The left limb can only be slightly extended (1 point); The left side is not as active and stretched as the right side (2 points); Bilateral forelimbs can be tested symmetrically (3 points). Climbing in the metal cage: None (0 points); Unable to mount (1 point); The left side is slightly weak (score 2 points); Can usually climb (3 points). Touch bilateral trunk reaction: none (0 points); No response on the left side (score 1 point); Weak left side reaction (score 2 points); The answer is symmetrical (3 points). Whisker reaction: None (0 points); No response on the left side (score 1 point); Weak left side reaction (score 2 points); The reaction is symmetrical (3 points).
Y-maze test
The Y-maze test was used to assess spatial learning and memory as described previously [15]. Experimental rats were placed in a Y-shaped maze (arm's length: 16 cm, arm width: 10 cm, height of the wall: 40 cm, Yihong Technology Co., Ltd., Wuhan, China) with three arms at 120° from each other and allowed to explore the three arms for 10 min freely. Before all experiments, the rats should be used for acclimatization for 2 days, during which the rats can visit all components freely for 10 minutes. The number of arm entries and alterations were recorded automatically using Smart Video Tracking Software 3.0 (Panlab, Barcelona, Spain). The percentage of spontaneous alternation was calculated as the number of correct alterations (number of total new arm entries), which is associated with the capacity of spatial short-term memory.
Novel object recognition test (NOR)
The novel object recognition test is widely used in the study of cognitive impairment[16]. First, to reduce stress levels, all groups of rats were placed in the experimental room and testing box for 10 min on the 2 successive days before the training phase. At 3 d post-MCAO, rats were trained to explore freely within a box (Yihong Technology Co., Ltd., Wuhan, China). The experimental method was described previously. The experiment was r recorded with a video camera (SNC-VB600B5, SSGE, Shanghai, China) cation placed on the old object. The exploration time ratio for novel objects during the testing phase was assessed for each group.
TTC staining
At 3 d after MCAO, TTC staining was used to detect the effect of TAT-PEP on cerebral infarction volume. The straightforward method is as follows[17]: The brain was removed under deep anesthesia and immersed in normal saline ice for 10 minutes. Put it into the cerebral sulcus mold, and the thickness of the brain slice is about 2 mm (cut in sequence along the coronal plane). Soak the brain slices in a 2% concentration of a 2,3,5-TTC solution with the same orientation, and incubate them in a constant temperature water bath (37 ℃, 30 min). After the staining effect is appropriate (the white area is the infarcted area, and the red area is the normal brain tissue area), transfer to 4% PFA for fixation overnight, take photos and analyze 24 hours later. Adobe photoshop cs3 image processing software was used to calculate the volume of cerebral infarction.
Magnetic resonance imaging (MRI) detected cerebral infarction volume.
At 3 d after MCAO, the small animal MRI imaging system was used to detect the effect of TAT-PEP on cerebral infarction volume. The straightforward method follows: After anesthesia, the rats were placed in a prone position with their heads in the center of the coil. The rapid acquisition relaxation enhancement sequence (along the coronal section) was used to scan with the visual intersection as the origin. The parameters are an echo time (TE) of 60 ms, a repetition time (TR) of 3000 ms, a thickness of 0.5 mm, and no spacing. The field of view is 2.56 cm × 2.56 cm. Acquire T2 weighted (T2WI) images, and then use image software for image analysis to measure the volume of ischemic injury area (i.e. abnormal high signal area), which is equal to the area of abnormally high signal area × the sum of slice thickness (plus spacing), was used to evaluate the cerebral infarction volume.
Nissl staining
Nissl staining (Beyotime Institute of Biotechnology, China) was performed to detect Nissl bodies in the cytoplasm of surviving neurons. At 3d after MCAO, Nissl staining was used to detect the effect of TAT-PEP on the survival of neurons in the penumbra. The methods are described as follows: Dry the slices at room temperature for 1 h, draw circles, and gently rinse them with 0.01M PBS 3 times (5 min/time). Add Nissl dyestuff (to filter in advance), and incubate at 37 ℃ for 20 min. Then gently wash off with pure water. The sections were dehydrated and transparent before using the neutral resin sealed. The microscope was used to observe and record in the open field. The integrated optical density/area of the staining in each group was acquired by 2 blinded investigators using ImagePro Plus 5.1 software (Media Cybernetics, Inc., Bethesda, MD).
FJC staining
At the 3d after MCAO, FJC staining was used to detect the effect of TAT-PEP on the number of degenerated neurons in the penumbra. The specific methods are as follows: Freeze the slices and dry them at room temperature for 1 h, draw circles, and gently rinse them with 0.01M PBS 3 times (5 min/time). Immerse the slices in a mixture of 1% sodium hydroxide and 80% anhydrous ethanol for 5 min. Then immerse it in 70% absolute ethanol, hydrate it for 2 min, and gently rinse it with deionized water 3 times (5 min/time). Incubate with 0.06% potassium permanganate for 10 min (shaking table at room temperature) and rinse with distilled water for 5 min (shaking table). Prepare 0.0001% FJC dye solution, and add acetic acid in the proportion of 1:1000. After dropping FJC dye solution, incubate it at 37 ℃ at constant temperature and away from light for 30 min, and gently rinse it with deionized water 3 times (1 min/time). Dry in the dark at room temperature, dehydrated with anhydrous ethanol for 2 min, make xylene transparent 3 times (2 min/time), and apply neutral gum film. The people who did not know the experimental group were observed by Olympus BX51 fluorescence microscope (excitation light wavelength was 450–490 nm), collected images, and recorded the number of FJC positive cells (FJC positive cells were green).
Immunofluorescence histochemical staining
On the 3rd day after MCAO, NeuN immunofluorescence histochemistry was used to detect the effect of TAT-PEP on the number of living neurons in the penumbra. The specific method is the same as that in Part I. First antibody (NeuN, 1:5000) was added, 4 ℃ overnight, 0.01M PBS solution was used for rinsing (5 min each time × 3 times), inject the secondary antibody (goat anti-mouse FITC, 1:5000), incubated at room temperature in the dark for 2 hours, rinse in the night for 3 times, observe and take photos under a fluorescence microscope after 50% glycerine film is sealed.
TUNEL staining
At the 3d after MCAO, TUNEL staining was used to detect the effect of TAT-PEP on the number of apoptotic neurons in the penumbra. The specific methods are as follows: The fixed brain tissue was immersed in 30% alcohol, embedded in paraffin, and sliced, with a thickness of 3 µ m. Paraffin sections were dewaxed with xylene for 5 min (2 min/time). After waxing, use a circle drawing brush to draw circles, drip protein kinase K cell permeating solution, incubate at 37 ℃ for 30 min, and then rinse gently with 0.01M PBS 3 times (5 min/time). Join 50 µ L DNase1 solution at room temperature for 10 min. After the slides are cleaned and dried, add the Tunel mixture. 37℃, 1 h. PBS cleaning again. Joining 30 µ L POD stops the reaction, and PBS has cleaned again. Drop the DAB color-developing solution at room temperature for 10min, then conduct PBS cleaning again. Drop hematoxylin for 30 s and gently wash it under pure water. Gradient dehydration, xylene transparent for 5min (twice), neutral resin film, open field microscope observation, and statistics of the number of Tunel positive cells (Tunel positive cells are brown black).
Electron microscope experiment
Experimental animals were anesthetized intraperitoneally with 0.3 ml/100g chloral hydrate (300 mg/kg), perfused and fixed with a solution containing 4% PFA and 0.05% glutaraldehyde, positioned on a stereotaxic device, and fixed for 2 hours. Coronal sections were made with a vibrating microtome with a thickness of 50 µ m and immersed in PBS solution containing 30% sucrose for 2 h. Dehydrated with gradient alcohol and Put the transparent continuous sections on copper mesh with supporting membrane, and observed the ultrastructure of neurons under the electron microscope.
Primary culture of cortical neurons
Primary cortical neurons were cultured as described previously[17]. SD rats who were pregnant 16.5–18.5 days (E 16.5–18.5 days) were dislocated and killed. A routine aseptic operation was carried out. Fetal rats were taken out and separated. In the D-Hank's solution (placed on ice), the fetal rat was decapitated with toothless ophthalmic tweezers, and the brain tissue was taken out. Under the microscope, the cerebral cortex was separated. The tissue was chylated through D-Hank's solution. Then trypsin (0.125%) was digested for 15 min (in a regular incubator at 37 ℃). Then, the tissue was carefully sucked out with an elbow dropper, added into a centrifuge tube (15 ml) containing DMEM solution of fetal bovine serum, and the digestion was stopped at room temperature for 5 minutes. Place the centrifuge tube in the centrifuge at 80 rpm for 5 min, discard the supernatant carefully, add 2 ml of DMEM solution containing fetal bovine serum, blow and repeatedly beat to prepare tissue and cell suspension, and use the cell filter (100 µ m) Filtering. After another centrifugation at 80 rpm for 5 min, it was added to the neuron culture medium (Neurobasal A, 2% B27, 1% glutamate, and 1% penicillin mixture). According to the experimental requirements, different-density cells were inoculated into 96 well plates, 24 well plates, or 6 well plates coated with poly-L-lysine (50 mg/mL) (Sigma, USA). These cells were grown in Neurobasal medium (Gibco, Invitrogen Corp, USA) supplemented with 2% B27, 1% glutamine, and 1% penicillin/streptomycin (Sigma, USA) at 37℃ under a humidified incubator in air containing 5% CO2. The purity of neurons was determined by immunocytochemistry for bIII-tubulin at 5-day after plated, which indicated that 95% of the cells in cultures were positive for βIII-tubulin (1:250; Millipore, Temecula, CA, USA) (data not shown)
Oxygen glucose deprivation model of neurons in vitro
The primary cultured cortical neurons were identified on the 7th day. Replace the culture medium (sugar-free and serum-free) and put it into the hypoxia constant temperature incubator (37 ℃, 5% CO2, 95% N2) for 1 h. Then carefully take out the culture dish or culture bottle, replace it with standard neuron culture medium (Neurobasal A, 2% B27, 1% glutamate, and 1% penicillin mixture), and place it in the traditional incubator (5% CO2, 21% O2, 37 ℃). After re-oxygenation and re-sugar, they were immediately given TAT-PEP or TAT mPEP. The experimental groups are as follows:
① MTT and LDH release tests [17] were used to detect the effect of TAT-PEP at different concentrations on the survival of neurons at 24 h after OGD. They were randomly divided into different groups (n = 6): Normal group, OGD group, OGD group, OGD + TAT mPEP (100 µg/L) group, OGD + TAT-PEP (50 µg/L) group, OGD + TAT-PEP (100 µg/L) group, OGD + TAT-PEP (200 µg/L) group.
② MTT and LDH release tests were used to detect the effect of TAT-PEP on neuronal survival after OGD (6h, 24h, 72h). They were randomly divided into different groups (n = 6): Normal group, OGD group, OGD + TAT mPEP (100 µg/L) group, and OGD + TAT-PEP (100 µg/L) group.
③ MDA level and SOD activity [18] was used to detect the effect of TAT-PEP on neuronal oxidative stress at 72 h after OGD. They were randomly divided into groups (n = 6): The normal group, OGD group, and OGD + TAT-PEP group (TAT-PEP was 100 µg/L).
④ TUNEL staining was used to detect the effect of TAT-PEP on neuronal apoptosis at 72 h after OGD. They were randomly divided into different groups (n = 6): The normal group, OGD group, and OGD + TAT-PEP group (TAT-PEP is 100 µg/L).
⑤ Western blot was used to detect the effect of TAT-PEP on the expression of apoptosis-related proteins at 72 h after OGD. They were randomly divided into different groups (n = 6): The normal group, OGD group, and OGD + TAT-PEP group (TAT-PEP is 100 µg/L).
Western blot
According to the time node of the experiment, the whole cell protein was extracted. The method follows: After washing PBS three times, add precooled Lysis cracking solution (100 µ L/well), carefully scrape the cells, and thoroughly mix the lysate with the cells. Transfer the suspension into the precooled EP tube, ice bath for 30 min, and centrifugation at 4 ℃ for 15 min, 12000 rpm. The total protein concentration of the tissues or cells was analyzed with a BCA kit (Sigma, CA, USA). Rabbit antibody against cleaved (active) caspase-3 (1:1,000; Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal antibodies against Bcl-2 or Bax (1:1,000, Santa Cruz, CA, USA), and β-actin (1:2,000, Anbo, USA). Subsequently, the blots were probed with horseradish peroxidase (HRP)-conjugated goat secondary antibody against rabbit or mouse IgG (1:1,000, Abcam, USA). Detection and quantitation were performed with a Typhoon 9400 Variable Mode Imager (GE Healthcare) and Lumi-Light Western Blotting Substrate (Roche Diagnostics) for HRP-labeled blots.
TUNEL staining
TUNEL staining was performed in vitro using an In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany). At 72 h after OGD injury, 4% PFA was added to fix at room temperature for 1 h. 0.01M PBS was gently rinsed 3 times (5 min/time), and then 0.3% hydrogen peroxide was used for 10 min. Add TUNEL reaction mixture for 1 h at 37 ℃ and stain with DAPI for 5 min at room temperature. Images were obtained with a microscope (BX60, Olympus). The integrated optical density/area of the positive TUNEL staining in each group was acquired as described above.
Statistical Analysis
All data are presented as the mean ± SD across the groups and were statistically analysed using GraphPad Prism 7.0 software (GraphPad Company, San Diego, CA, USA). Continuous data were tested for normal distribution and analysed by one-way ANOVA (followed by Tukey’s multiple comparisons tests) or Kruskal–Wallis test (followed by Dunn’s multiple comparisons tests). Two-way ANOVA was applied to analyse the neural dendritic complexity. P value of less than 0.05 was considered statistically signifcant.