动物造模 z-bio 2022-07-12 425

　　Introduction: Propofol is the most commonly used intravenous anesthesia for 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%. Operating room and post-anesthesia ICU accounted for 55%, and severe hypoxemia with oxygen saturation<81% in 20% and 13%, respectively. Apnea induced by propofol anesthesia without supplemental oxygen can lead to a gradual decrease in blood oxygen saturation, further hypoxia, and even death. We have previously shown that propofol by itself or hypoxia by itself does not directly induce significant neuronal apoptosis. However, propofol-induced respiratory depression can produce lower blood oxygen concentrations in air or under mild hypoxic conditions, leading to neuronal degeneration. We also found that propofol itself induced short-term cognitive impairment; however, propofol combined with hypoxia severely impaired cognitive function in neonatal rats. In addition, propofol can lead to impaired 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 delivery to study the effects of propofol anesthesia on spatial memory, neuronal apoptosis, and long-term enhancement of hippocampal CA-1 area in juvenile rats.

　　Animals: 7-day-old juvenile 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), room air (propofol group-air PA), or 18% oxygen (propofol group-hypoxia, PH), intraperitoneal injection of 50 mg/kg Propofol. Control rats were exposed to 50% oxygen (control-oxygen CO). Room air (control group - air, CA) or 18% oxygen (control group - hypoxia, CH) and the same volume (ip) of fat emulsion (5.0 ml/kg) were injected intraperitoneally. The anesthesia group or the control group were injected once a day for 7 consecutive days. Blood oxygen saturation (%) and respiratory rate (RR) were monitored throughout.

　　Immunohistochemistry: 24 hours after the last injection, 6 pups were randomly selected from each group. Immunohistochemistry of choice for activation of caspase-3. Anesthetized animals were perfused with 10 U/ml heparinized saline. Subsequently, 4% formaldehyde in PBS is perfused, procedures that preserve neuronal antigenicity and prevent tissue liquefaction. After rat brain removal, hippocampal CA1 was isolated and divided into small pieces. Small pieces were fixed in paraformaldehyde, embedded in paraffin, and then cut into coronal sections with 5 μm thick sections. These paraffin sections were sequentially used for deoxynucleotidyl transferase dutp nick end labeling (TUNEL) and immunohistochemistry. Tissue sections were deparaffinized in xylene, then rinsed with graded ethanol, and then antigen was recovered in sodium citrate buffer in a microwave oven at 100°C for 10 min and cooled to room temperature. Experiments were performed using a tissue staining kit. The color reaction was carried out with 3,30-diaminobenzidine. The primary antibody used was at a 1:200 dilution for activation of caspase-3. Counterstained with hematoxylin and fixed with resin. TUNEL staining was performed based on terminal deoxynucleotidyl transferase-mediated in situ nicked end labeling of deoxyuridine triphosphates according to the instructions of the TUNEL kit.

　　Field excitatory postsynaptic potential recordings and LTP induction success rate checks: 24 hours after the last injection, animals were sacrificed under isoflurane (2%) and nitrous oxide (N2O, 70%) anesthesia. The heart was perfused with 20 ml of cerebrospinal fluid. Place the brain rapidly in slicing liquid at 0–4°C oxygen saturation for 1–2 min. We cut out 400-micron-thick bilateral hippocampal brains by vibratome slices and placed the slices in a mixture (in mM) containing the following: 124 NaCl, 26NaHCO3, 1.25 NaH2PO4, 2.8 KCl, 2 CaCl2, 2 MgSO4, and 10 Glucose, oxidized with 95% O2 and 5% CO2. Gas was continuously bubbled into the mixture and incubated at 35°C for 30–45 min. 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 a rate of 1.5 ml/min at 35°C. A bipolar tungsten stimulating electrode (0.01 mm in diameter, 10 μm spacing) was placed in the hippocampal CA3 area. The stimulation intensity was 0.1-0.25 mA with a pulse width of 0.5 ms. We placed glass recording electrodes into the CA1 region radiata to record electrical stimulation-induced field excitatory postsynaptic potentials (field excitatory postsynaptic potentials or FEPSPs). Samples were processed and stored using PCLAMP 9.2 software.

　　Detection of the success rate of LTP induction: The stimulation intensity with the maximum response value induced by FEPSP of 50% was selected as the basic stimulation, and high-frequency LTP-induced electrical stimulation was given after the basis was stable for 30 minutes. After high-frequency electrical stimulation, the fEPSP slope increased by more than 20% and lasted for more than 30 minutes. It is considered that the LTP induction was successful. 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 between each group is 30s.

　　Morris Water Maze: The Morris water maze test includes the Position Navigation Test (PNT) and the Spatial Probe Test (SPT). We tested each mouse's ability to acquire spatial learning and spatial memory using a positional navigation test and a spatial probe test (n = 8). The water maze experimental design 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 movements of each rat and analyzed the data obtained using the MWM motion detection software.

　　Positional Navigation Test (PNT): On postnatal day 28, the day before the formal MWM test, we put the rats into the swimming pool for 2 minutes for an acclimation test. Rats received 4 acquisition trials (T1-T6) per day for 6 consecutive days. In the field test, rats were placed in one of four virtual quadrants of the swimming pool, facing the wall, and randomly placed in the swimming pool. Before the next trial, the rats were allowed to stand on the platform for 60 s using a trial interval. In each trial, we used a computerized tracking system to measure swimming speed, time spent (escape latency), and distance traveled to the platform (escape path length). Escape latency was recorded as 120 seconds. We placed animals that did not find the platform within 120 seconds on the escape platform.

　　Space Probe Test (SPT)

　　To examine spatial reference memory, a probe test was performed 24 hours after the last training session. During probe testing, we removed the platform from the pool to measure the spatial deviation of previous platform positions. We placed each rat in the quadrant opposite the training platform position, followed the rat for 120 s, and measured the percentage of time spent in the previous target quadrant and the number of intersections at the previous platform position.

　　Conclusion: Propofol induces respiratory depression: monitor blood oxygen saturation and respiratory rate during propofol anesthesia. All propofol-treated animals had symptoms of respiratory distress and significantly lower respiratory rates compared to controls. Sao2 levels in propofol-treated animals were significantly reduced when exposed to air and mild hypoxia. Interestingly, when propofol-treated animals were supplemented with oxygen, there were no significant changes compared to control animals. The results showed that propofol induced respiratory depression and subsequent hypoxia in the air environment.

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

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

　　Decreased fEPSP and LTP success rates under hypoxic conditions: Field excitatory postsynaptic potentials: There were no significant differences in fEPSP amplitudes between CO, CA, and CH groups at the same electrical stimulation intensity. Compared with the control group, the fEPSP amplitude of the propofol group was decreased, and the decrease of the fEPSP amplitude in the PA and PH groups was significantly higher than that in the PO group. fEPSP decreased in PO, PA and PH groups. There were no significant differences among the three control groups.

　　LTP success rate: The fEPSP slope before high-frequency stimulation was defined as the reference value. In the control group, fEPSP was significantly enhanced after HFS, and the success rate of LTP induction was 60%. The results showed that fEPSP was significantly enhanced in the treatment group, and the LTP success rate after HFS was about 43%. There was no significant change in EPSP in PA group and PH group, and the successful induction rate of LTP was about 31% and 22%, respectively. Compared with the control group, the LTP success rate of propofol-treated rats was decreased. The successful induction rate of LTP in the PA and PH groups was lower than that in the PO group.

　　Effects 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 in the PO group was decreased, and the mean escape latency was significantly prolonged. Compared with the CA group, the average escape time of the PA group was longer and the number of platform location crossings was reduced. 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 PO group, PA group and PH group had longer escape latency and decreased platform crossing times. There were no significant differences between the CO, CA and CH groups.

　　Conclusion: In conclusion, propofol anesthesia itself can lead to short-term impairment of cognitive function, but propofol combined with hypoxia can lead to long-term impairment of cognitive function in neonatal rats. The effects of repeated propofol anesthesia on LTP were more pronounced, with further learning and memory impairments longer under air conditions than under supplemental oxygen conditions. Conclusions: Hypoxia causes cognitive impairment in neonatal rats after anesthesia, indicating the importance and necessity of oxygen supply during anesthesia to prevent postoperative cognitive impairment in patients, especially children.