动物造模 z-bio 2022-09-02 429

　　1 Materials and methods

　　1.1 Materials

　　1.1.1 Experimental animals

　　10 healthy clean-grade female Sprague-Dawley (SD) rats, 4 weeks old, were provided by the Laboratory Animal Center of Central South University, with a body weight of 70-90 g entering the laboratory. General health requirements: bright hair color, no depilation, no disease in the limbs and tail, and no skew in the neck. All rats were kept in the same animal room 1 week before the experiment to adapt to the environment, and they could drink water and eat freely, and the food was standard pelleted feed. The temperature of the animal room was controlled at 20-22 °C, and the ventilation was good to maintain the natural light-dark biological rhythm.

　　1.1.2 Feed and main equipment and chemical reagents

　　The standard pelleted feed for rats was provided by the Department of Laboratory Zoology, Central South University; the high-fat feed was self-made (the formula was taken from Zhang Tao et al.: ordinary feed 50%, lard 12%, milk powder 10%, soybean meal 5%, peanut 2.5% , sucrose 5%, rapeseed oil 1%, salt 1%); the open field experiment box and supporting Supermaze software are products of Shanghai Xinruan Information Technology Co., Ltd.; Leica cryostat is a product of Wuhan Zhongji Scientific Instrument Co., Ltd.; Nikon microscope is Nikon Instrument Co., Ltd.; paraformaldehyde is a product of Tianjin Kemeiou Chemical Reagent Co., Ltd.; sodium dioxyphosphate is a product of International Group Chemical Reagent Co., Ltd.; disodium hydrogen phosphate is a product of Changsha Antai Fine Chemical Industry Co., Ltd.; sucrose is a product of Guangdong Products of Guanghua Technology Co., Ltd.; 0.01 mol/L phosphate energy buffer solution and DAB chromogenic reagent are products of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; c-Fos primary antibody is a product of Santa Cruz Company, USA, goat serum, secondary antibody and tertiary antibody It is a product of Vector Labs in the United States; anhydrous ethanol and xylene are products of Sinopharm Chemical Reagent Co., Ltd.

　　1.2 Methods

　　1.2.1 High-fat diet model establishment

　　Ten female rats were randomly divided into two groups of 5 rats in each group. The control group was fed with ordinary feed for 15 weeks, and the high-fat group was fed with self-made high-fat feed for 15 weeks. All rats had free access to food and water during the modeling period, and the food and water were changed every 2 days.

　　1.2.2 Behavioral testing

　　1.2.2.1 Open field experiment

　　Open field experiment box (length × width × height: 100 cm × 100 cm × 50 cm), the bottom of the box is marked with 25 equal squares (20 cm × 20 cm), and the 16 squares along the four walls are called peripheral grids, The remaining 9 squares are the central squares. During the experiment, the rats were placed in the central grid, and the number of the rats passing through the central grid, the total number of grids passing through, and the stay time in the central grid were recorded for 5 minutes. The percentage of time spent in the central grid in the total time (5 min) was negatively correlated with anxiety-like emotions in rats. The total number of grid crossings reflects the motor function of the animal.

　　1.2.2.2 Observation of non-competitive predatory behavior

　　The predation model refers to the method of Li et al.: the observation of animal predation was carried out in a separate, quiet and soundproof room with an open field (length × width × height: 150 cm × 150 cm × 50 cm), The bottom and four walls of the open field are black. Before the observation of predatory behavior, the rats to be tested need to go on a diet for 1 week, and be given a small amount of food and drink enough water every day to reduce their body weight to about 80% of the previous one; d Fasting (without water) for 12 h, the rats were placed in the open field at 4:00 pm on the day of observation, and after 2 h of adaptation, a plastic rat cage (length × width × height: 30 mm) was placed on the side of the open field. cm × 18 cm × 16 cm), put 250 g of standard rat pellet feed on the barbed wire top cover of the rat cage, and after 2 h, collect the remaining feed on the top cover of the rat cage and the feed scattered in the open field. , and weigh them separately. The amount of food scattered in the open field is the amount of predation by the rats.

　　1.2.2.3 Observation of competitive predatory behavior

　　The observation equipment was used to observe non-competitive predatory behavior. Fasting (without water) for 12 hours before the observation, the rats were placed in the open field at 4:00 pm on the day of observation, and after 2 hours of adaptation, a plastic bag was placed on the side of the open field A rat cage (length × width × height: 30 cm × 18 cm × 16 cm) was made, and a rat of the same sex as the rat to be tested, from different litters, and never contacted before, was placed in the rat cage. 250 g of standard rat pellet feed was placed on the barbed wire top cover of the rat cage. After 2 h, the feed scattered in the open field and the remaining feed on the top cover of the rat cage were collected and weighed respectively. The amount of food scattered in the open field is the amount of predation by the rats.

　　1.2.3 Immunohistochemical detection of c-Fos protein in rat anterior cingulate cortex When rats were preying on noncompetitively for 1 h and 45 min, the rats were quickly taken out and treated with 10% oxyaldehyde (40 mg/kg) in After anesthesia by intraperitoneal injection of rats, the brains were quickly taken out, and the brain tissue was placed in fixative solution and fixed overnight, and then placed in 15% and 30% sucrose PBS solution and soaked in sugar to the bottom, and the optimal cutting temperature (optimal cutting temperature, OCT) complexes were embedded and placed in a constant temperature cryostat for serial sections with a thickness of 30 m, and the sections containing the anterior cingulate cortex were taken. The sections were stained with c-Fos immunohistochemical staining and observed by Olympus microscope. Three sections were selected from the control group and the high-fat group, and the positive cells were counted under a 10×10 magnification microscope.

　　1.3 Statistical processing

　　The data were expressed as mean ± standard deviation (x ± s), and GraphPad Prism 5.0 software was used for statistical analysis. Group t test was used for comparison between the two groups, and P<0.05 was considered to be statistically significant.

　　2 Results

　　2.1 Behavioral testing

　　2.1.1 Open field experiment

　　The results of the open field experiment are shown in Figure 1: There was no significant difference in the percentage of residence time in the central grid between the high-fat group (10.43%±2.87%) and the control group (12.23%±2.79%) ( t = 0.4 50, P = 0.6647); the total number of grids passed, the high fat group ( 127.10% ± 37.89% ) and the control group ( 98.90% ± 17 ) . 15 % ), the difference was not statistically significant (t=0.676, P=0.5172).

　　2.1.2 Predatory behavior

　　The results of predatory behavior are shown in Figure 2: in the observation of non-competitive predatory behavior and competitive predatory behavior, the predation amounts of rats in the control group were (84.80±2.77) g and (89.94±1.96) g per 2 h, respectively. . In the observation of non-competitive predation and competitive predation in the high-fat group, the predation amounts per 2 h were (13.85±1.08) g and (12.25±1.07) g respectively; compared with the corresponding control group, the predation amount decreased ( Non-competitive predatory behavior t=21.65, P<0.001; competitive predatory behavior t=32.17, P<0.001).

　　2.2 The immunohistochemical detection of c-Fos protein in the anterior cingulate cortex of rats is shown in Figure 3: The number of positive cells in the control group was (88.17 ± 4.14) cells/field, and the number of positive cells in the high-fat group was (45.00±2.56) cells/field, the number of positive cells in the control group was higher than that in the high-fat group (t=8.868, P<0.001).

　　3 Discussion

　　Predatory behavior is a highly comprehensive model that can be used to discuss different issues in psychology, physiology, neurobiology, etc. At the same time, predatory behavior is also an important model for theoretical research in behavioral science. Predatory behavior is related to decision-making, effort Cognitive functions such as effort and cost/benefit estimation are closely related. Using predatory behavior to elucidate the effect of long-term high-fat diet on cognitive function, and to explore the effect and mechanism of long-term high-fat diet on rats' predatory behavior, has guiding significance for people's health.

　　Before many researchers use rodent predatory behavior to explore the neurobiological mechanism of cognitive function, animals often need to be artificially trained. Excessive training affects the behavioral autonomy of rats, thereby affecting the results of predatory behavior. reflect its true neurobiological nature. Therefore, the author used the laboratory rat predation behavior observation model of Li et al. to exclude the influence of human factors on the predation behavior of rats, and quantitatively measured the predation behavior of rats to explore its neurobiological mechanism.

　　The open field test is a classic method for evaluating motor function and state anxiety in rodents. We use this to determine that animal predatory behavior disorder is not caused by changes in motor ability, exploratory behavior, and animal emotions. The results of the mine field experiment between the control group and the high-fat group showed that there was no significant difference between the two groups, suggesting that the high-fat diet had no effect on the motor function of the rats.

　　In this study, it was found that the rats in the high-fat group had significantly less predation than the control group in non-competitive predatory behavior and competitive predatory behavior, which suggested that the rats in the high-fat group were reluctant to make efforts to obtain more food, indicating that high Fatty diet does affect predatory behavior in rats.

　　Multiple brain regions and various chemicals in the central nervous system are involved in the regulation of rodent predatory behavior. The anterior cingulate cortex is located in the medial part of the prefrontal cortex and is involved in the regulation of complex behaviors such as cognition, learning, and social assessment. Individual primates with damaged anterior cingulate cortex fail to properly integrate risk and reward in reinforcement-mediated choice tasks. Research on the damage of the anterior cingulate cortex in rodents shows that the rats with damaged anterior cingulate cortex give up the behavior of "getting more for more" and are more willing to spend less effort to get less income. Studies by Li Mingbo et al. showed that the predation behavior of rats with bilateral anterior cingulate cortex damage was significantly impaired, and the amount of competitive predation decreased most significantly. The results of this study showed that the amount of predation in the high-fat group was significantly reduced, suggesting that the predation disorder in the high-fat diet rats may be related to the functional changes of the anterior cingulate cortex. As a third messenger involved in intracellular signal transduction, the expression of c-Fos protein can reflect the activity of neurons to a certain stimulus, and is often used as a marker of neuronal activation. In this study, the expression of c-Fos in the anterior cingulate cortex was significantly reduced in the high-fed rats during the predation process, suggesting that the mechanism of long-term high-fat diet affecting the predatory behavior of rats may be related to the decline of neuronal function in the anterior cingulate cortex. The mechanism still needs to be further explored.