动物实验 z-bo 2020-08-13 876

　　The open field experiment and the study of the effects of the Morris water maze on the emotional behavior and learning and memory of rats have attracted the attention of scholars at home and abroad. Clinical studies have found that recurrent febrile seizures in the early years have a certain relationship with later life; experiments have confirmed that febrile seizures can cause neuronal degeneration and necrosis in the hippocampus of immature brain, and pathological ultrastructural changes of organelles and synapses. The author established a rat model of febrile convulsions by using a hot bath method to explore the effects of febrile convulsions on the emotional behavior, learning and memory ability of rats.

　　1　Object and method

　　1.1 Subject 36 male SD (Sp raque 2 Dawley) rats aged 21 days (provided by the Experimental Animal Center of Xi'an Jiaotong University School of Medicine), weighing (50 ± 5) g; random numbers were equally divided into febrile seizure group (FS ), fever control group (FG), normal control group (NG), the number of rats in each group is 12; each group is raised in a standard environment with free eating and drinking.

　　1.2　Method

　　1.2.1 Establishing a febrile convulsion model using a hot water bath method to establish a febrile convulsion rat model: put the rats in the FS group into a transparent glass tube (diameter 10 cm, height 50 cm), and there are multiple The small hole communicates with the outside; put the tube upright in a constant temperature (45.0±0.25) ℃ water bath (100 cm×70 cm×50 cm), and adjust the water in the tube by placing a rubber gasket on the bottom of the glass tube The height of the rat is based on the fact that the rat is only exposed above the neck when standing along the wall of the tube; each rat uses this method to induce convulsions once in the morning and afternoon, and a total of 10 times for 5 consecutive days; observe the occurrence of convulsions and record the occurrence of convulsions The incubation period (the time interval from the rat entering the water to the start of the convulsion, the recording unit is min), grade, and the duration (the time interval from the start of the rat convulsion to the end of the convulsion, the recording unit is seconds). Rats in F G group were bathed in hot water for 2 minutes in the same way without seizures. Group N G did not perform any treatment. The level of convulsion refers to the standard of W Jiang et al.: Grade 0 means no convulsions; Grade 1 means facial twitching; Grade 2 means nodding; Grade 3 convulsions; Grade 4 means general rigidity; Grade 5 means general tonic clonus.

　　1. 2. 2　open field test (OFT) 　Model: XR-XZ301 (developed by Shanghai Xinruan Information Technology Co., Ltd.) Divide the bottom of a 60 cm×60 cm×60 cm topless wooden box into 36 equal parts In a small square, the rat that has completed 10 convulsions is placed in the center of the bottom of the wooden box, and the score of the number of squares passed by the rat's activity within 5 minutes, the number of hindlimb standing times and the number of feces discharged are recorded. The scoring standard is: 1 point for rats with more than 1/2 of the body entering the adjacent grid, 1 point for standing hindlimbs, and the total is the total score. The study believes that the score of the number of grids passed reflects the excitability of the animal; the number of hindlimb standing represents the ability of the rat to adapt to the unfamiliar environment; the number of feces excreted indicates the degree of tension in the rat.

　　1. 2. 3 　 Passive avoidance test (PAT) 　 SF K2 Ⅰ dark avoidance test box (made in Japan) has two distinct chambers, one is bright and one is dark, and there is a small door in between. The dark habit makes the rat instinctively choose to enter the dark room, Rats entering the dark room were immediately shocked (3 mA, 50 Hz) and passively escaped back to the safe bright room. If you do not enter the dark room for 5 minutes, it is considered to have completed the study. Record the number of times that the rat entered the dark room by mistake before completing the study after suffering 10 febrile seizures, which mainly reflects the rat's recent learning ability; put it back into the bright room again after 24 hours , Record the time when the rat enters the dark room from the bright room, which is the memory latency, which mainly reflects the ability of the rat's recent memory.

　　1. 2. 4　Morris water maze test (Morris water maze test, MWM T) 　Morris water maze test model: XR-XM101 (developed by Shanghai Xinruan Information Technology Co., Ltd.) consists of a circular pool (diameter 130 cm, height 50 cm), Camera and operation analysis system (microcomputer and software) are composed. The circular water surface is divided into 4 quadrants by two imaginary vertical lines passing through the center of the circle. The day before the test, the rats were allowed to swim freely in the pool for 2 minutes to familiarize themselves with the environment. The test time is 4.5 days. A circular transparent platform (10 cm in diameter and 28 cm in height) is placed in the middle water area of one of the quadrants. The platform is hidden 2 cm below the water surface and the water temperature is kept at about 24℃. The test is carried out 4 times in the morning and afternoon each day, and each rat randomly enters the water from the midpoint of each quadrant to the wall once. After entering the water, the rat tries its best to escape the flood, and when it finds the platform, it lives on it. The image acquisition system and analysis The system automatically records the escape latency for the rat to find the platform. If the platform is not found in 120 seconds, the operator will lead the rat to the platform and rest for 4 seconds, and record the incubation period as 120 seconds. The average value of each rat's 4 times in the morning and afternoon was used as a period value (sectio n, I2Ⅸ) for analysis. The effect of open field experiment and Morris water maze on emotional behavior, learning and memory in rats

　　1.3　Statistical processing 　All measurement data are expressed in...x ±s. The analysis of variance was carried out according to the nature of the data, and the test level was α = 0.05.

　　2　结 　果

　　2.1 Occurrence of febrile seizures 　The FS group all induced febrile convulsions, and a total of 120 convulsions were induced, and no one died. The number of convulsions (and percentages) of grades 5, 4, 3, 2, and 1 were 89 (74.0%), 5 (4.6%), 20 (16.5%), 4 (3.5%), 2 (1.4%) times; the incubation period of convulsions was (4.02±0.76) min; the duration of convulsions was (1.01±0.53) min.

　　2.2 　 Changes in emotional behavior in the open field test The FS group has the lowest tracing score (37.48±7.09), and the number of hindlimb standing (12.11±4.41) is the least, within 5 minutes The number of fecal pellets excreted (10.62±2.32) was the most; the FS group compared with the NG and FG groups in the crossing score (q=4.72, 4.18; P<0.01), hindlimb standing The number of times (q=5.12, 4.85; P<0.01) and the number of fecal pellets (q=5.33,5.02; P<0.01) were very significant. In addition, in terms of the number of feces excreted, the difference between the N G and F G groups is also very significant (q=3.17, P<0.05).

　　2.3 Changes in learning and memory in the dark avoidance test FS group rats made the most mistakes (4.02±1.29) when they reached the level of learning, and the difference was very significant compared with NG and FG groups (q= 6. 2 4, 5. 3 1; P<0.01). After 24 h, the memory latency of rats in the FS group (169.71±103.22) was the shortest, which was significantly different from those in the N G and F G groups (q = 5.69, 5.28; P <0.01). The results show that repeated febrile seizures can impair the short-term learning and memory ability of rats.

　　2.4 Changes in spatial learning ability in the Morris water maze test The ability of rats in the FS group to find hidden platforms in the Morris water maze is manifested as a significantly prolonged escape latency, suggesting that repeated febrile seizures can impair the spatial learning ability of rats; variance Analysis shows that the FS group has a very significant difference with the NG and FG groups in terms of escape latency (F= 152.46, P<0.01)

　　3 Discussion

　　febrile seizures are one of the most common emergencies in pediatrics. The clinical manifestations at the time of the attack are sudden and severe, which have adverse effects on family members and society. Whether febrile seizures can damage children's emotional behavior, learning and memory in the future has attracted scholars' attention.

　　3.1 The scientific nature of this animal model of febrile seizures This experiment used a hot bath method to establish a febrile seizure model and modified the experimental equipment, so that all rats in the febrile seizure group induced convulsions of different levels. The previous literature reported that rats The susceptibility to drowning has improved. The improvement of the experimental equipment (glass cylinder with an empty bottom) also makes it difficult for the rat's feces to be discharged into the water bath, thus ensuring the cleanliness and sanitation of the experimental operation. Compared with other models, the establishment of a febrile seizure model is an ideal experimental method.

　　3.2 The effect of febrile seizures on affective behavior and learning and memory. This experiment found that the excitability of rats in the FS group decreased in the open field test, and the ability to adapt to the new environment decreased, and at the same time, they were prone to nervous adverse reactions; the results of the dark avoidance test It shows that repeated febrile seizures can reduce the short-term learning and memory ability of rats. The Morris water maze is an ideal device designed by British psychologist Morris to reflect the spatial learning and memory abilities of small rodents. It can be used as a comparative study on the impairment of human learning and memory abilities by diseases or stress. The escape latency of rats in the FS group in the Morris water maze was significantly prolonged, reflecting the impaired spatial learning ability of rats. The above results indicate that repeated febrile seizures can lead to impaired emotional behavior, learning and memory in rats, similar to previous studies.

　　The possible mechanism of

　　3.3　 febrile seizures affecting affective behavior and learning and memory is unknown. The mechanism of repeated febrile seizures leading to impairment of emotional behavior and learning and memory in rats is unknown. Previous studies have found that repeated febrile seizures can cause pathological changes in the structure of the hippocampus, including neuronal degeneration and necrosis in the hippocampus, changes in synaptic morphology (abnormal widening of the synaptic gap), and abnormal mossy fibre sprouts in the hippocampus. routing), and the resulting abnormal synaptic connections, abnormal distribution and expression of NMDA (N2 methyl 2D aspartate) receptors closely related to learning and memory, and abnormal signal transduction pathways. The hippocampal structure belongs to the ancient cortex and is an important part of the limbic system. The loop composed of the hippocampus and the prethalamic nucleus, the lamina nucleus, the hypothalamic papilla thalamic tract, and the cingulate is an important neuroanatomical basis for regulating behavior, learning and memory, and emotional response. Therefore, the above findings about the changes in hippocampal structure and function may be an important part of explaining the impairment of emotional behavior and learning and memory ability in rats caused by repeated febrile seizures. However, the relationship between these processes and the degree of impact of each process on the impairment of brain function need to be further studied.