Animals and Groups
30 SPF male SHR rats aged 10 weeks, animal license number: SCXK (Beijing) 2016-0011, weighing 180g-220g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Place it in a standard 12h: 12h light-dark environment, maintain a room temperature of 20-25 ℃, relative humidity (50 ± 10)%, free drinking and eating. Turn on the lights at 7:00 in the morning and turn off the lights at 19:00 at night. ZT0 is defined as the time when the lights are turned on. Before entering the experimental protocol, all rats were fed continuously for 1 week to minimize stress to ensure consistency. In the experiment, the rats were randomly divided into 2 groups: SHR group, SHR+tMCAO group, 15 rats in each group. The protocol used in this study and the animal ethics involved have been approved by the Animal Ethics Committee of Xuzhou Medical University.
Experimental Reagents and Instruments
DEM animal physiological signal telemetry system (DSI, American); microplate reader, electrophoresis instrument, film transfer instrument (Bio-Rad, American); gel imaging analysis system (ProteinSimple, American). Rabbit anti-mouse CRY1, PER1, CLOCK and BMAL1 antibodies (Abclonal, China). FITC labeled goat anti-rabbit IgG (Shanghai Aibixin Biotechnology Co., Ltd.); BCA kit (Shanghai Biyuntian Biotechnology Co., Ltd.). PVDF membrane 0.45 μm (Santa Cruz, USA); ECL chemiluminescence kit (Shanghai Aibixin Biotechnology Co., Ltd.); PER1, BMAL1, CLOCK, CRY1 Elisa kit (Wuhan Yiaibo Company).
Ambulatory Blood Pressure Measurement
A blood pressure remote sensing monitoring system was established. The rats were anesthetized with 1% sodium pentobarbital (0.4 ml/100 g) intravenously, and an incision of about 2 to 3 cm was made at the midline of the abdomen to separate the abdominal aorta. Clamp the blood vessel temporarily, insert the needle into the artery, and insert the implant catheter into the abdominal aorta about 5~6 mm (against the direction of blood flow) along the hole. Fix the catheter with fiber sheet and adhesive glue, and then flush the blood vessel with 2% lidocaine to avoid vasoconstriction and spasm. The intestine segment is returned to the abdominal cavity, and the implant body is fixed on the abdominal wall and the abdomen is closed. After the operation, the rats were reared in a single cage and moved freely without restraint. After 7 to 10 days, the blood pressure and heart rate became stable. The cage was placed on the receiving board to collect the data, which was converted by the signal processor and analyzed by relevant software to monitor the changes in blood pressure. Since the circadian rhythm of rats is opposite to that of humans, the rate of blood pressure drop is (average night blood pressure-average day blood pressure)/average night blood pressure × 100%, which is a quantitative index to judge the circadian rhythm of blood pressure.
Preparation and Verification of Rat tMCAO Model
SHR was anesthetized with 10% chloral hydrate (3.5mL/kg) intraperitoneally. Routinely disinfect the neck skin, cut along the midline of the neck. Separately expose the left common carotid artery, left internal carotid artery, and left external carotid artery. The vascular clamp temporarily clamps the left internal carotid and common carotid artery, and advances the thread tether through the incision on the left external carotid artery and inserts it into the left internal carotid artery until a slight resistance is felt. After 2 hours, the thread plug was withdrawn, and the cerebral blood flow began to reperfusion spontaneously. The operation of the rats in the sham operation group was similar to that of the ischemia-reperfusion group, but no thread plug was inserted into the left internal carotid artery. All experimental animals eat and drink freely after being awake. Nerve injury behavior score (Longa classification method) was used to score the two groups of rats. The specific scoring principles are as follows: 0 points: no defect; 1 point: the contralateral forelimb cannot be fully extended; 2 points: turning to the opposite side while walking; 3 points: falling to the opposite side while walking; 4 points: unable to walk spontaneously. TTC staining was used to evaluate the degree of cerebral infarction, red is normal brain tissue, and white is post-infarction brain tissue.
Elisa Detects the Content of Serum Clock Protein
Take blood samples every 3h. Fix the rat in a fixed rat cage, expose the tail of the rat outside the cage, and place it in hot water at 40℃ for 5-10s. Sterilize the tail with an alcohol cotton ball, then pierce the intravenous needle a few centimeters up from the tip of the tail and pull it out immediately. Take 0.3 mL of blood each time, centrifuge at 4000 rpm for 15 minutes, take the serum and store in the refrigerator at -80℃. Take out the kit to equilibrate at room temperature for 30 minutes, and at the same time take out the blood sample and place it at room temperature. First configure the standard: Take 150μL of the standard and add 150μL of the standard diluent, and dilute 5 times in sequence. Add 50μL of the standard and 40μL of the sample to each reaction well. Make a duplicate hole for the standard and 3 holes for the sample. Add 10 μL of antibody to the sample wells. Add 50μL of streptavidin-HRP to the standard and sample respectively, cover with sealing film, shake gently to mix, and incubate at 37°C for 1h. When the time is up, carefully uncover the sealing film, discard the liquid, and spin dry. Add 200μL of washing solution to each well, let it stand for 30s and discard it, repeat this 4 times, and pat dry. Then add 100 μL of color developing solution to each well, gently shake and mix, incubate at 37°C for 30 minutes in the dark, and then add stop solution. Measure the absorbance of each well at 450nm wavelength with a microplate reader, and process the data with Excel.
Statistical Methods
Continuous variables are expressed as mean±standard deviation (Mean±SD). Use one-way analysis of variance to compare the differential expression of proteins between groups. The relationship between the two variables uses Pearson correlation analysis. Two-sided test P <0.05, indicating that there are statistical differences between the two groups. All statistical analyses were performed using SPSS 26.0.