动物造模,丙泊酚 z-bo 2020-09-09 529

　　Introduction: Propofol is the most commonly used intravenous induction and maintenance anesthesia. Hypoxemia is a common complication during anesthesia. In a previous clinical study, the incidence of mild hypoxemia (Spo2 86-90%) was 53%. The operating room and post-anaesthesia ICU accounted for 55%, respectively, and severe hypoxemia with blood oxygen saturation<81% was 20% and 13%, respectively. The apnea caused by propofol anesthesia without supplemental oxygen will cause the blood oxygen saturation to gradually decrease, further hypoxia, and even death. We have previously demonstrated that propofol itself or hypoxia itself did not directly induce significant neuronal apoptosis. However, the respiratory depression caused by propofol can produce lower blood oxygen concentration in the air or under mild hypoxic conditions, leading to neuronal degeneration. We also found that propofol itself induces short-term cognitive impairment; however, the combination of propofol and hypoxia severely impairs the cognitive function of neonatal rats. In addition, propofol can cause damage to LTP. LTP, the mechanism involved in memory formation has been extensively studied in the hippocampus. However, the effect of propofol on LTP under hypoxic conditions is unknown. In this study, we imitated clinical medication and oxygen to study the effects of propofol anesthesia on spatial memory, neuronal apoptosis, and long-term enhancement of hippocampal CA-l area in young rats.

　　Animals: 7-day-old young SD rats (12-16g) were used. 84 rats were randomly divided into 6 groups (n=14), propofol or control group. Rats in the propofol group. Exposure to 50% oxygen (propofol group-oxygen, PO), indoor air (propofol group-air PA), or 18% oxygen (propofol group-hypoxia, PH), intraperitoneal injection of 50 mg/kg Propofol. The rats in the control group were exposed to 50% oxygen (control group-oxygen CO). Indoor air (control group-air, CA) or 18% oxygen (control group-hypoxia, CH) and intraperitoneal injection of the same volume (ip) fat emulsion (5.0 ml/kg). The anesthesia group or the control group were injected once a day for 7 consecutive days. Both blood oxygen saturation (%) and respiratory rate (RR) are monitored throughout the process.

　　Immunohistochemistry: 24 hours after the last injection, 6 pups were randomly selected from each group. Select immunohistochemistry to activate caspase-3. The anesthetized animals were infused with 10 U/ml heparin saline. Subsequently, perfusion of PBS containing 4% formaldehyde, these procedures can protect the antigenicity of neurons and prevent tissue liquefaction. After the rat’s brain was taken, the hippocampus CA1 was separated and divided into small pieces. The small pieces are fixed in paraformaldehyde, embedded in paraffin, and then cut into crowns with a section of 5 μm thick. These paraffin sections were used in turn for deoxynucleotidyl transferase dutp nick end labeling (TUNEL) and immunohistochemistry. The tissue sections were deparaffinized in xylene, then rinsed with gradient ethanol, then the antigen was recovered in sodium citrate buffer in a microwave oven at 100°C for 10 minutes and cooled to room temperature. Use tissue staining kit for experiment. Use 3,30-diaminobenzidine for color reaction. The main antibody used is used to activate caspase-3 in a 1:200 dilution. Counterstain with hematoxylin and fix with resin. According to the instructions of the TUNEL kit, TUNEL staining was performed based on in situ deoxyuridine triphosphate nicked end labeling mediated by terminal deoxynucleotidyl transferase.

　　field excitatory postsynaptic potential recording and LTP induction success rate check: 24 hours after the last injection, the animals were killed under anesthesia with isoflurane (2%) and nitrous oxide (N2O, 70%). The heart is perfused with 20ml of cerebrospinal fluid. Quickly place the brain in the slice liquid with blood oxygen saturation of 0–4°C for 1–2 minutes. We sliced out 400-micron-thick bilateral hippocampal brains with a vibrator, and placed the slices in a mixture containing the following substances (unit: mM): 124 NaCl, 26NaHCO3, 1.25 NaH2PO4, 2.8 KCl, 2 CaCl2, 2 MgSO4 and 10 Glucose is oxidized with 95% O2 and 5% CO2. Gas is continuously bubbled into the mixture and incubated at 35°C for 30–45 minutes. After the incubation period, the sections were placed at room temperature for 1 h.

　　field excitatory postsynaptic potential recording: hippocampal slices were continuously perfused with oxygen saturation recording solution at 35°C at a rate of 1.5 ml/min. Place bipolar tungsten stimulation electrodes (with a diameter of 0.01 m m and a spacing of 10 μm) into the hippocampal CA3 area. The stimulation intensity is 0.1-0.25 mA, and the pulse width is 0.5 ms. We put the glass recording electrode into the CA1 area radiation layer to record the field excitatory postsynaptic potential (field excitatory postsynaptic potential or FEPSP) induced by electrical stimulation. Use PCLAMP 9.2 software to process and save the samples.

　　LTP induction success rate detection: The stimulus intensity with the maximum response value of 50% induced by FEPSP is selected as the basic stimulus, and high-frequency LTP-induced electrical stimulation is given after the basic is stable for 30 minutes. After high-frequency electrical stimulation, the slope of fEPSP increased by more than 20% and lasted more than 30 minutes. It is considered that LTP was successfully induced. There are two sets of high-frequency electrical stimulation parameters. The frequency is 100Hz, the pulse width is 0.5ms 100 pulses, and the interval for each group is 30s.

　　Morris water maze: Morris water maze test includes position navigation test (PNT) and space probe test (SPT). We use the position navigation test and the spatial probe test to test the ability of each mouse to acquire spatial learning and spatial memory (n=8). The design of the water maze experiment is a stainless steel circular water tank (100 cm in diameter and 50 cm in depth) filled with water (24±0.5°C). We used a video tracking system to record the swimming actions of each rat, and analyzed the data obtained using the MWM motion detection software.

　　Position Navigation Test (PNT): On the 28th day after delivery, the day before the official MWM test, we put the rats in the swimming pool for 2 minutes for an adaptation test. Rats received 4 collection tests (T1-T6) per day for 6 consecutive days. In the field test, rats were placed in one of the four virtual quadrants of the swimming pool, facing the wall, and randomly placed in the swimming pool. Before the next test, the rats were allowed to stand on the platform for 60 s using the test interval. In each test, we use a computer tracking system to measure swimming speed, time spent (escape latency), and distance traveled to the platform (escape path length). The escape latency was recorded as 120 seconds. We put the animals that did not find the platform within 120 seconds on the escape platform.

　　Space Probe Test (SPT)

　　In order to check the spatial reference memory, a probe test was performed 24 hours after the last training. During the probe test, we removed the platform from the pool to measure the spatial deviation of the previous platform position. We placed each mouse in the quadrant opposite the training platform location, followed the mouse for 120 seconds, and measured the percentage of time spent in the previous target quadrant and the number of intersections on the previous platform location.

　　Conclusion: Propofol causes respiratory depression: monitor blood oxygen saturation and respiratory rate during propofol anesthesia. Compared with the control group, all propofol-treated animals had symptoms of respiratory distress and the respiratory rate was significantly reduced. When propofol-treated animals were exposed to air and mild hypoxia, the level of Sao2 in their bodies was significantly reduced. Interestingly, when propofol-treated animals were supplemented with oxygen, there was no significant change compared to control animals. The results show that propofol can cause respiratory depression and subsequent hypoxia in an air environment.

　　Propofol induces neuronal apoptosis in air and mild hypoxia: In our experiments, propofol caused a significant increase in brain cell apoptosis under air and mild hypoxia. There were fewer TUNEL positive cells in the CO, CA, CH, and PO groups, and the apoptotic index was significantly increased in the PA and PH groups. The apoptosis index of pulmonary artery and pulmonary artery in PH group was significantly higher than that in CA and CH groups (P<0.05), and the average value of TUNEL index in PA and PH group was higher than that in PO group (P<0.05).

　　caspase-3 expression: caspase-3 expression in the hippocampus of PA and PH groups was significantly up-regulated. Compared with the control group and the PO group, the immunohistochemistry of the PA group and the PH group was significantly enhanced.

　　The reduction in the success rate of fEPSP and LTP under hypoxic conditions: field excitatory postsynaptic potential: under the same electrical stimulation intensity, there was no significant difference in fEPSP amplitude in the CO, CA and CH groups. Compared with the control group, the fEPSP amplitude of rats in the propofol group was reduced, and the reduction of fEPSP amplitude in the PA and PH groups was significantly higher than that in the PO group. The fEPSP of PO, PA and PH groups decreased. There was no significant difference between the three control groups.

　　LTP success rate: The fEPSP slope before high-frequency stimulation is defined as the reference value. In the control group, fEPSP was significantly enhanced after HFS, and the success rate of LTP induction reached 60%. The results showed that fEPSP in the treatment group was significantly enhanced, and the success rate of LTP after HFS was about 43%. The EPSP of the PA group and the PH group did not change significantly, and the successful induction rate of LTP was about 31% and 22%, respectively. Compared with the control group, the success rate of propofol treatment of LTP in rats was reduced. The successful induction rate of LTP in PA and PH groups was lower than that in PO group.

　　The effect of propofol exposure on long-term spatial learning and memory under hypoxic conditions: Compared with the CO group, the RR on the first and second days of the PO group was reduced, and the average escape latency was significantly prolonged. Compared with the CA group, the PA group had a longer average escape time and reduced the number of platform crossings. Compared with the CH group, the average escape latency was longer, and the number of platform crossings was significantly reduced in the PH group. Compared with the PO group, the PA group and the PH group had longer escape latency and reduced the number of platform crossings. There is no significant difference between CO, CA and CH groups.

　　Conclusion: To sum up, propofol anesthesia itself can cause short-term impairment of cognitive function, but propofol combined with hypoxia can cause long-term impairment of cognitive function in neonatal rats. Repeated propofol anesthesia has a more significant effect on LTP, and further learning and memory impairment under air conditions are longer than those under supplemental oxygen conditions. It is concluded that hypoxia causes cognitive impairment in neonatal rats after anesthesia, which illustrates the importance and necessity of oxygen supply during anesthesia to prevent postoperative cognitive dysfunction in patients, especially children.