慢性间歇低氧动物模型 z-bo 2020-10-13 363

　　【Abstract】 Objective To establish a rat model of chronic intermittent hypoxia and study its spatial learning and memory ability and the changes of neuron ultrastructure in hippocampal CA1 area. Methods 40 SD pups of 3 to 4 weeks old who were successfully trained in the eight-arm maze were randomly divided into a 2-week intermittent hypoxia group (2H), a control group (2C) and a 4-week intermittent hypoxia group (4H) and a control group (4C) Establish an animal model of chronic intermittent hypoxia, and eight-arm maze test after the end of intermittent hypoxia

　　Learning and memory ability, transmission electron microscope observation of the ultrastructural changes of neurons in hippocampal CA1 area. Results Intermittent hypoxia group reference memory errors [(3. 30 ± 1. 06) times, (2. 00 ± 0.67) times], working memory errors [(2. 30 ± 0.67) times, (1. 00 ± 0.82) times] and the total number of errors

　　[(5.40 ± 1.78) times and (3.00 ± 0.82) times] were higher than those of the control group (P<0.05), and with the prolongation of intermittent hypoxia time, the number of such errors increased (P < 05); The morphology of nerve cells in the CA1 area of hippocampus and the organelles in the cytoplasm of the intermittent hypoxia group under the electron microscope have obvious changes. in conclusion

　　Chronic intermittent hypoxia can cause a decline in spatial learning and memory in young mice, which may be related to the changes in the ultrastructure of neurons in the CA1 region of the hippocampus.

　　Obstructive sleep apnea hypopnea syndrome (OSAHS) is one of the common diseases in children, with an incidence of about 1% to 10%. OSAHS has a great impact on children's body systems, especially the nervous system. The damage caused by OSAHS can lead to learning and memory dysfunction in children, but the pathophysiological mechanism of OSAHS causing learning and memory damage is not completely clear. Therefore, if the pathophysiological process of OSAHS and the impairment of brain learning and memory function can be simulated, and its pathogenesis can be elucidated from the cellular and molecular level, it will provide a theoretical basis for the prevention and treatment of learning and memory impairment in children with OSAHS. According to the pathophysiological characteristics of chronic intermittent hypoxia in children with OSAHS during sleep, this experiment establishes a chronic intermittent hypoxia rat model and evaluates the physiological indicators of its cardiovascular system. The eight-arm maze test is used to determine its spatial memory, to study OSAHS and learning The relationship and mechanism of memory.

　　Materials and Methods

　　One, materials

　　50 healthy male SPF SD (Spra2gue Dawley) pups (3 to 4 weeks old) with a body weight of 80-100 g, provided by the Experimental Animal Center of Wenzhou Medical College, certificate number: [SYXK (Zhejiang) 200520061] ; CYES2Ⅱ oxygen and carbon dioxide gas analyzer (Shanghai Jiading Xuelian Instrument Factory); POW ERLAB/8SP physiological signal acquisition and analysis system (Australia AD Instruments); Intermittent hypoxia rat stainless steel breeding cabin and air simulation control cabin are self-made; air Compression pump; solenoid valve; single chip microcomputer; medical compressed oxygen (concentration> 99.5%) and compressed high-purity nitrogen (concentration> 99.99%), provided by Wenzhou Medical Oxygen Factory; LKB2088 ultra-thin microtome; H27500 Transmission electron microscope; eight-arm maze (provided by Shanghai Xinruan Information Technology Co., Ltd.); bait for maze (Brain and Intelligence Research Center, Zhejiang University School of Medicine).

　　2. Method

　　1. Eight-arm maze test: This experiment refers to Zou LB and other methods for training. Before training, hungry the young mice to about 85% of their original body weight, and then keep this body weight during training. Then adapt to the maze for 2 d,

　　Once a day. During adaptation, the two young rats were placed in the maze at the same time, free to move and ingest the bait for 15 minutes. Perform 2 times/d training after adaptation. In each training session, only four of the eight arms were placed with bait (arms 3, 5, 6, and 8 respectively), and this order was maintained throughout the experiment. The young rats are placed in the central area of the maze and covered with a small plastic bucket. After 15 seconds, the plastic bucket is lifted to release the young rats. The young rats can choose to enter any arm to take the bait. The baby mouse enters the arm with bait and takes the bait for a correct choice, otherwise it is a wrong choice.

　　Re-entering the bait arm is called a working memory error (working memory error, WME), the first time you enter the arm without bait is called a reference memory error (reference memory error, RME), the sum of the two is the total memory error (total memory error, TE). The number of wrong selections for 5 consecutive trainings is ≤1, and the WME must be zero at the same time, and the training is considered successful.

　　2. Grouping: Take 40 pups that have been successfully trained in the maze, and randomly divide them into a 2-week hypoxia group (2H), a 4-week hypoxia group (4H), a 2-week control group (2C), and a 4-week control group (4C). 10 in each group.

　　3. Animal model establishment: OSAHS animal model establishment is improved by referring to McGuire M, Gozal D and other methods: gas cylinders, pressure reducing valves, flow meters, control programs, relays, solenoid valves, displays, etc. constitute a control system, and self-made intermittent hypoxia chambers The main body is made of stainless steel, 125 cm * 48 cm * 24 cm in size. The middle part of the hatch cover is made of glass, which is convenient for observation; there are 4 check valves for air intake and 4 check valves for exhaust on the side wall. The internal pressure can always maintain normal pressure. During the experiment, 4 squirrel cages were built in, and 4 trays covered with straw paper were placed on the bottom to receive excrement. The young rats moved freely without restriction on food.

　　Different gas sources have different ventilation pipes, all of which are controlled by a program-controlled solenoid valve. Nitrogen is supplied at a pressure of 0.3 KP for 30 s, 30 s is stopped, and oxygen is supplied at a flow rate of 25L/min for 12 s, and it is stopped for 18 s. This is one cycle. Every day 7.5 hours (8: 50 ~ 16: 20). During the cessation of ventilation, the connected oxygen meter measures the high value, low value and carbon dioxide concentration of the intermittent hypoxia chamber. The diagram of the oxygen concentration change curve in the chamber is shown in Figure 1. The control group was placed in an air simulation control cabin under the same conditions, with compressed air and controlled by the same single-chip computer program.

　　During the experiment, each cabin was covered with a thick cloth to keep the cabin dark to simulate night sleep. After leaving the cabin every day, the pups were placed under fluorescent lights to simulate the daytime, without restrictions on activities and diet. Keep the young rats hungry before the end of the experiment, and test the maze on the day the hypoxia ends. The reference object and the food arm are the same as the training process during the whole measurement process.

　　4. Animal treatment: the day after hypoxia is over, pentobarbital sodium (35mg/kg-1 bw, ip) anesthesia, right heart catheterization method to measure pulmonary artery pressure, physiological recorder records pulmonary artery pressure wave, and calculates the average pulmonary artery pressure (mPAP) ), after the left common carotid artery was intubated to measure the carotid artery pressure (mCAP), 1 pup was randomly selected from each group, fixed on the back, the thoracic cavity was opened, the heart was exposed, and the needle tube precooled to 0 ~ 4 ℃ physiological saline was sucked Puncture the left ventricle, cut open the right atrial appendage, quickly infuse about 100ml of normal saline, and then slowly infuse with about 200mL of 4% glutaraldehyde precooled to 0～4 ℃, then quickly take the brain, and use the routine electron microscope procedure The specimens were made, 2.5% glutaraldehyde pre-fixed, 1% osmium acid post-fixed, embedded in Epon812, sectioned with LKB2088 ultrathin microtome, double stained with lead 2 uranium, and observed by H27500 transmission electron microscope. Each young mouse cuts off the right atrium tissue, cuts the right ventricle along the edge of the ventricular septum, weighs the right ventricular free wall (RV) and left ventricle plus ventricular septum (LV + S) respectively, and calculates RV / (LV + S) The weight ratio is used as an indicator of right ventricular hypertrophy.

　　5. Statistical methods: Data is expressed as x ±s, and SPSS12.0 statistical software is used for statistical processing. For comparison between multiple groups, such as normal distribution and homogeneity of variance, one-way analysis of variance and LSD2 t test were used, and Tamhane’s T2 test was used for uneven variance.

　　Result

　　One. Changes in pulmonary artery pressure, carotid artery pressure and right ventricular weight ratio

　　Table 1 shows that the mPAP of the rats in the 2H group is 18.72% higher than that in the 2C group (P<0.05), and the mPAP of the rats in the 4H group is 16.87% higher than that in the 4C group (P<0.05). there="" was="" no="" significant="" difference="" in="" mpap="" between="" the="" two="" hypoxic="" groups="" and="" control="" p="">0.05). There was no significant difference in mCAP, RV/LV + S between the groups (P>0.05).

　　Two, eight-arm maze test results

　　Table 2 shows that RME, WME and TE of 2H group are higher than those of 2C group (P<0.05); RME, WME and TE of 4H group are higher than those of 4C group (P<0.05); RME, WME and TE of 4H group are higher than those of 2C group (P<0.05). Group 2H (P<0.05) p="">0.05).

　　3. Ultrastructure observation of hippocampal CA1 area

　　2C and 4C groups have normal neurons, mitochondria, synapses, myelinated nerve fibers, and clear nuclear membranes. In the 2H group, the nuclear membrane of nerve cells became blurred, deeply stained, and slightly pyknotic, and the mitochondria became slightly vacuolated. In the 4H (Figure 3A, B) group, the nuclear membrane of nerve cells is obviously blurred, the boundary is unclear, the chromatin becomes darker, the mitochondrial cristae is slightly dilated, and focal myelination is widespread.

　　Discuss

　　In children, untreated OSAHS can cause many complications, such as growth delay, mental retardation, high blood pressure, pulmonary hypertension, right heart failure, and learning and memory impairment. The relationship between OSAHS and children's learning and memory disorders has gradually received widespread attention at home and abroad in recent years, but there is little research on it. The main pathophysiological process of OSAHS is the collapse of the upper airway during night sleep, which causes repeated hypoxia-reoxygenation cycles and sleep. Structural disorder. Some people think that the damage to the nervous system caused by chronic intermittent hypoxemia at night may be an important mechanism leading to the impairment of learning and memory in children with OSAHS. Therefore, we simulated its pathophysiological process and made a chronic intermittent hypoxia rat model for research.

　　In the experiment, we evaluated the physiological indicators of the cardiovascular system of the model, and found that the rats in the intermittent hypoxia group of 2 weeks and 4 weeks had increased pulmonary artery pressure, which was consistent with similar models at home and abroad, and indirectly proved that this model was modeled. success.

　　The radial eight-arm maze (four arms with food) is a classic method for measuring animal spatial memory. It can detect the reference memory and working memory of the animal at the same time. The animal learns and remembers itself and the bait by observing the fixed reference objects around it. The relative position of the white snack pill at the end of the maze arm. Under the experimental conditions, the number of incorrect selections (including RME, WME, and TE) of the pups in the 2H and 4H groups in the radial eight-arm maze was higher than that in the 2C and 4C groups, which confirmed that intermittent hypoxia can cause the young mice to learn and memory capacity. (Including working memory and reference memory) damage. In addition, this experiment also showed that the RME, WME, and TE of the 4H group were higher than those of the 2H group, while the difference between the 4C and 2C groups was not statistically significant, indicating that this kind of spatial learning and memory impairment is aggravated with the extension of intermittent hypoxia. .

　　The hippocampus is recognized as a brain area closely related to spatial learning and memory. The CA1 area of the hippocampus is particularly sensitive to hypoxia and ischemia. By inhibiting the apoptosis of hippocampal neurons, it can improve the learning and memory impairment caused by hypoxia and ischemia. In this experiment, an electron microscope was used to observe the CA1 area of the hippocampus of experimental young rats. Death phenomenon. This ultrastructural change affects the neuronal energy metabolism and other normal physiological activities, so that the hippocampal synaptic plasticity of young rats is affected, and this plasticity is the neural basis of learning and memory; in addition, due to repeated hypoxia -Reoxygenation leads to oxidative stress, which leads to the overproduction of ROS and participates in nerve cell apoptosis. Therefore, we speculate that the mechanism of chronic intermittent hypoxia causing space learning and memory impairment in young mice may be related to the damage of hippocampal CA1 area and early neuronal apoptosis caused by oxidative stress. Other molecular biological mechanisms need to be further studied.

　　This experiment uses the chronic intermittent hypoxia baby mouse model to verify the clinical phenomenon that OSAHS causes learning and memory dysfunction in children, and through the observation of the ultrastructure of the hippocampus, it preliminarily explains the possibility of learning and memory dysfunction caused by human OSAHS from a pathological point of view. The mechanism suggests that the importance of effective early treatment of OSAHS in children and the intervention and regulation of neuronal apoptosis may become a new approach for OSAHS treatment.