动物实验,高脂饮食 z-bo 2020-08-19 515

　　As the pace of life accelerates, people’s dietary structure changes. The intake of a large number of high-fat foods can not only cause a series of health problems, such as obesity, cardiovascular disease, metabolic syndrome, diabetes, etc.; it can also lead to People's neuropsychiatric abnormalities, such as can damage the memory function of mice, cause increased activity and anxiety and depression behavior in mice; it can also cause cognitive impairment in mice. There are fewer studies on the predatory behavior of high-fat diets. The predatory behavior of rats involves their handling and hoarding of food and competition with other rats. It is to search for more food while meeting current needs. Satisfying future use is a strategy for adaptation and survival. Therefore, this experiment will observe the effect of long-term high-fat diet on the predatory behavior of rats, and observe the changes in the expression of c-Fos protein in the anterior cingulate cortex of the rat brain to explore the effect and mechanism of high-fat diet on the predatory behavior of rats.

　　1 Materials and methods

　　1.1 Material

　　1.1.1 Experimental animals

　　10 healthy and clean female Sprague-Dawley (SD) rats, 4 weeks old, provided by the Experimental Animal Center of Central South University, weighing 70-90 g into the laboratory. Whole body health requirements: bright hair color, no hair loss, no disease of limbs and tail, no crooked neck. All rats were bred in the same animal room one week before the start of the experiment to adapt to the environment, and they were able to drink and eat freely, and the food was standard granular feed. The temperature of the animal room is controlled at 20-22 ℃, well ventilated, and the natural light-dark biological rhythm is maintained.

　　1.1.2 Feed and main equipment and chemical reagents

　　The standard granular feed for rats is provided by the Department of Laboratory Animals of Central South University; the high-fat feed is homemade (the formula is taken from Zhang Tao, etc.: ordinary feed 50%, lard 12%, milk powder 10%, soybean meal 5%, peanut 2.5% , Sucrose 5%, rape oil 1%, table salt 1%); 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 Instruments Co., Ltd.; Nikon microscope is a Nikon Instrument Co., Ltd. product; paraformaldehyde is a product of Tianjin Komiou Chemical Reagent Co., Ltd.; sodium dioxophosphate 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 color reagent are products of Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; c-Fos primary antibody is a product of Santa Cruz of the United States, goat serum, secondary antibody and tertiary antibody It is the product of American Vector Labs; the absolute ethanol and xylene are the product of Sinopharm Chemical Reagent Co., Ltd.

　　1.2 Method

　　1.2.1 High-fat diet model establishment

　　Randomly divide 10 female rats into two groups, each with 5 rats. The control group was fed with ordinary feed for 15 weeks, and the high-fat group was fed with homemade high-fat feed for 15 weeks. All rats eat and drink freely during the model building, and the feed and water are changed every 2 days.

　　1.2.2 Behavioral testing

　　1.2.2.1 Open field test

　　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 the outer grids. The remaining 9 squares are central squares. During the experiment, place the rat in the center grid, take a video and record the number of the central grid, the total number of grids and the stay time of the central grid for 5 min. The percentage of staying time in the central grid to the total time (5 min) was negatively correlated with anxiety-like emotions in rats. The total number of grids crossing reflects the movement function of the animal.

　　1.2.2.2 Observation of non-competitive predation behavior

　　The predation model refers to the method of Li et al.: The observation of animal predation behavior is 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, give a small amount of food a day, and drink enough water to reduce their body weight to about 80%; then 1 before the observation. d Fasting (without water) for 12 hours, put the rat in the open field at 4:00 pm on the day of observation, and after adapting for 2 hours, put a plastic squirrel cage (length×width×height: 30 cm×18 cm×16 cm), put 250 g of standard rat pellets on the top cover of the squirrel cage. After 2 hours, collect the remaining feed on the top cover of the squirrel cage and the feed scattered in the open field. , And weigh them separately. The amount of feed scattered in the open field is the predation of rats.

　　1.2.2.3 Observation of competitive predation behavior

　　Observation equipment and non-competitive predation behavior observation, fasting (can not help water) for 12 hours before observation, put the rat in the open field at 4:00 pm on the day of observation, after acclimatization for 2 hours, put a plastic on the open field side To make a squirrel cage (length×width×height: 30 cm×18 cm×16 cm), a rat that has the same sex as the rat to be tested, comes from a different litter, and has never been in contact before. Put 250 g of standard rat pellets on the top cover of the squirrel cage. After 2 h, collect the feed scattered in the open field and the remaining feed on the top cover of the squirrel cage and weigh them separately. The amount of feed scattered in the open field is the predation of rats.

　　1.2.3 Immunohistochemical detection of c-Fos protein in the anterior cingulate cortex of rats. When the rats were predating non-competitively for 1 h and 45 min, the rats were quickly taken out and treated with 10% oxyaldehyde hydrate (40 mg/kg). After intraperitoneal injection of anesthesia, the brain was quickly taken out of the rat, the brain tissue was placed in the fixative solution and fixed overnight, and then placed in 15%, 30% sucrose PBS solution to soak the sugar to the bottom, using the optimal cutting temperature (optimal cutting temperature, The OCT) complex was embedded and placed in a constant temperature cryostat for continuous slices with a thickness of 30 m. The slices containing the anterior cingulate cortex were taken. The sections were stained by c-Fos immunohistochemical staining and Olympus microscope observation using immunohistochemical ABC method. The control group and the high-fat group each took 3 slices, took pictures and counted the positive cells under 10×10 magnification.

　　1.3 Statistical processing

　　The data are expressed as mean±standard deviation (x±s), and GraphPadPrism 5.0 software was used for statistical analysis. The group t test was used for comparison between the two groups, and the difference was considered statistically significant with P<0.05.

　　2 Results

　　2.1 Behavioral testing

　　2.1.1 Open field test

　　The results of the open field experiment are shown in Figure 1: The percentage of residence time in the central grid, the high-fat group (10.43%±2.87%) compared with the control group (12.23%±2.79%), the difference was not statistically significant (t = 0.4 50, P = 0.6647); the total number of grids that travelled, 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 predation behavior are shown in Figure 2: In the observation of non-competitive predation behavior and competitive predation behavior of the control group, the predation amount per 2 h was (84.80±2.77) g and (89.94±1.96) g, respectively . In the observation of non-competitive and competitive predatory behavior in the high-fat group, the predation amount per 2 h was (13.85±1.08) g and (12.25±1.07) g, respectively; compared with the corresponding control group, the predation amount was reduced ( 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 rat anterior cingulate cortex is shown in Figure 3: The number of positive cells in the control group is (88.17 ± 4.1 4) per field of view, and the number of positive cells in the high fat group is (45.00±2.56)/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 explore different issues in many disciplines such as psychology, physiology, neurobiology, etc. At the same time, predatory behavior is also an important model for theoretical research in behavioral science. Predatory behavior and decision-making, effort Cognitive functions such as effort and cost/benefit estimation are closely related. Using predatory behavior to clarify the effect of a long-term high-fat diet on cognitive function, and to explore the effect and mechanism of a long-term high-fat diet on the predatory behavior of rats is of guiding significance to people's health.

　　Before many researchers use rodent predation behavior to explore the neurobiological mechanism of cognitive function, they often have to conduct artificial training on animals. Excessive training affects the behavioral autonomy of rats, which affects the results of predatory behavior. Reflect its true neurobiological nature. Therefore, the author used Li et al.'s laboratory rat predatory behavior observation model to eliminate the influence of human factors on the predatory behavior of rats, and quantitatively measured the predatory behavior of rats to explore its neurobiological mechanism.

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

　　This study found that the predation of the high-fat group rats in non-competitive and competitive predatory behaviors was significantly less than that of the control group. This suggests that the high-fat group rats are unwilling to make efforts to obtain more food, indicating that the high-fat group Fat diet does affect the predatory behavior of rats.

　　Multiple brain regions of the central nervous system and a variety of chemical substances are involved in regulating the predatory behavior of rodents. The anterior cingulate cortex is located in the medial part of the prefrontal lobe and participates in the regulation of complex behaviors such as cognition, learning, and social evaluation. Primates with damaged anterior cingulate cortex cannot properly integrate risk and reward in the reinforcement-mediated selection task. Research on damage to the anterior cingulate cortex in rodents shows that rats with damaged anterior cingulate cortex give up the behavior of "paying more and getting more", and are more willing to spend less effort to earn less income. Li Mingbo and other studies have shown that the predatory behavior of rats after bilateral anterior cingulate cortex is damaged is significantly impaired, and the decrease in competitive predation is the most obvious. The results of this study showed that the predation of rats in the high-fat group was significantly reduced. It is speculated that the predatory behavior disorder of high-fat diet rats may be related to the changes in the anterior cingulate cortex. As the third messenger involved in intracellular signal transduction, c-Fos protein expression can reflect the activity of neurons to a certain stimulus, and is often used as a sign of neuron activation. In this study, the expression of c-Fos in the anterior cingulate cortex of rats on a high diet was significantly reduced during predation, 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 further discussion.