动物模型 z-bo 2020-07-25 763

　　Mainly focus on establishing exercise models and studying the effects of exercise on physiology and pathology, in order to promote the science of sports training, improve athletic performance and the role of exercise therapy in disease rehabilitation. Treadmills are increasingly used in experimental research. . Reasonable and effective applications are also tools for studying exercise and metabolism, exercise and cardiovascular diseases, cerebrovascular diseases, and neurological diseases.

　　1. The purpose of the experiment

　　Use sound, light and electricity to stimulate rats to run on a treadmill to achieve their training goals.

　　2. Experimental principle

　　The main part of the treadmill is the conveyor belt. The surface material helps the animal grasp. The back wall of the channel is equipped with stimulation electrodes, acoustic devices and bulbs, and the stimulators of each channel are independent of each other. If the animal refuses to run, or the running speed is lower than the experimental requirement, then retract it to the conveyor belt and touch the stimulator on the back wall. Due to strong electrical or acoustic stimulation, the animal runs at the speed of a treadmill.

　　3. Experimental materials and methods

　　(1) Experimental materials

　　1. Experimental equipment, animal experiment treadmill. 2. Experimental animals and experimental groups (1) Rats or mice (in this experiment, rats are taken as examples).

　　(2) According to the purpose of the experiment, the rats are divided into groups, usually a training group and a quiet control group. Training groups can also continue to be divided according to different training intensities, and each rat is numbered.

　　(3) Raised in another cage, free food, animal room temperature 21-25℃, relative humidity 40%-60%, circadian rhythm changes of natural light.

　　(2) The specific method of the experiment

　　(1) Turn on the treadmill and its corresponding software, place the mouse in the corresponding track according to the number, and then close the lid. (2) Set the corresponding parameters, such as time, speed, electrical stimulation intensity, and then click [Start].

　　(3) After training, remove the rat and switch to another group of animals. The other steps are the same as above.

　　(4) Training 5-6 days a week. All arrangements are in the evening.

　　(3) Precautions for experimental operation

　　(1) When selecting rats with similar weight, there is no significant difference in the physiological indicators of rats with similar weight. Weigh once a week and observe changes in body weight.

　　(2) Before formal training, a period of adaptive training is required to allow the rats to have time to adapt.

　　(3) Please use the treadmill carefully to avoid accidents, raise the treadmill when the rat’s tail is clamped, and disinfect with iodophor after running. (4) Excessive stimulation can cause physiological changes in rats, including increased adrenaline. Therefore, in the treadmill experiment, especially in the case of electrical stimulation, the intensity and frequency of stimulation should be reduced as much as possible. The electrical stimulation is usually adjusted to 0.5-0.8 mA and less than 1 mA.

　　(5) Rats consume more food during exercise, so they need to supplement food and water, and replace litter in time.

　　(6) According to the mouse's lifestyle, training should be carried out at night as much as possible.

　　Auxiliary experiment: The effect of treadmill training on the recovery of neurological function and the expression of synaptophysin in rats with cerebral ischemia

　　an experimental purpose

　　Observed the changes of neuronal recovery and synaptic element expression in rats with cerebral ischemia during treadmill training.

　　2. Experimental principle

　　Many studies have shown that exercise training can promote the recovery of nerve function after cerebral hemorrhage. In this experiment, the neurological function score (mNSS) was used to observe the accelerating effect of treadmill training on the neurological recovery of rats with cerebral hemorrhage. Western blot detection is used to explain the role of synaptophysin in the recovery process of rats.

　　3. Experimental materials and methods

　　(1) Experimental materials

　　(1) Equipment required: animal experiment execution platform, sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE).

　　(2) Reagents needed: RIPA lysate, synaptophysin antibody (from rabbit), secondary antibody. (II) The specific method of the experiment

　　1. Experimental animals and group of 24 healthy adult male SD rats, weighing (250±20) g, 7 weeks old (provided by the Experimental Animal Center of Kunming Medical University). They were randomly divided into sham operation group, ischemia control group and exercise training group, with 8 animals in each group. The rats are housed in separate cages and eat freely. The animal room temperature is 21-25°C, the relative humidity is 40%-60%, and the circadian rhythm of natural light changes. 2. Model preparation: SD rats were anesthetized by intraperitoneal injection of 3.6% chloral hydrate (1ml/100g) and then fixed. The Longa external carotid artery suture was used to prepare the right middle cerebral artery occlusion (MCAO) model. In the sham operation group, a thread plug was also inserted, but the depth was less than 1 cm, and the blood flow of the middle cerebral artery was not blocked. After establishing the model, 20,000 U of gentamicin was injected intraperitoneally within 3 days to prevent infection. The experimental animals were scored for neurobehavior 24 hours after surgery, and animals with scores of 2-4 were included in the experiment.

　　3. After the treadmill training, the sports training group received 3d adaptive training on the 4th day at 10m/min per week, 30min per day, 10m/min, 20min per day. The 20-day formal training starts. We will conduct training from 6pm to 8pm on Friday, and there will be a total of 4 weeks of vacation on weekends. The sham operation group and the ischemia group received no exercise training. 4. Neurological function score 24 hours after surgery, 4, 8, 12 and 20 days to evaluate the nervous system severity score (mNSS) of the three groups of rats. The final score is 4 items, including motor, sensory and reflex test, motor test, sensory test, average beam test, comprehensive evaluation score of the severity of neurological damage caused by loss of reflex and abnormal movement is 13-18), moderate injury The score is 7-12 points, and the minor injury score is 1-6 points).

　　5. Western blotting for each group Rats were anesthetized with sodium pentobarbital, decapitated, and the cortex and hippocampus of the right brain were excised, and stored at -80°C for western blot detection. Lyse the tissue with RIPA lysis buffer. After homogenization in a water bath, it was sonicated. Centrifuge at 12000r/min for 15 minutes at low temperature, and collect the supernatant. The BCA method determines the concentration of protein samples. Take 15μg protein sample, add 5μl loading buffer, and boil it at 100°C for 5 minutes. 100g/LSDS-PAGE75V, protein separation for 2 hours. Transfer to PVDF membrane (Millipore) for 2 hours at 350 mA. The skim milk made with TBST was sealed for 2 hours. Synaptic primary antibody (rabbit source, 1:2000) was diluted in TBS and incubated overnight at 4°C. The membrane was rinsed 3 times with TBST for 10 minutes. Dilute the secondary antibody with TBS (goat anti-rabbit, 1:8000) and incubate for 2 hours at room temperature. The membrane was rinsed 3 times with TBST for 10 minutes. Create colors and images by using a chemiluminescent gel imaging system. Using β-actin as an internal control, images were acquired after imaging, the optical density value was measured, and the relative expression of the protein was reflected by the ratio of the target band to the optical density of β-actin for comparison between groups. I went.

　　(3) Precautions for experimental operation

　　(1) Choose rats of the same weight so that the test results will not change much.

　　(2) Before formal training, a period of adaptive training is required to allow the rats to have time to adapt.

　　(3) Please use the treadmill carefully to avoid accidents, raise the treadmill when the rat’s tail is clamped, and disinfect with iodophor after running. (4) Excessive stimulation can cause physiological changes in rats, including increased adrenaline. Therefore, in the treadmill experiment, especially for electrical stimulation, the stimulation intensity and frequency should be as low as possible. 0.8mA, maximum 1mA or less.

　　(5) Rats will consume more food during exercise, so they should add food and water in time.

　　(4) Experimental results obtained

　　(1) Compared with the ischemia control group and the exercise training group, the sham operation group has basically no neurological deficits. Twenty days after surgery, the neurological function of the ischemic control group and the exercise training group recovered significantly. The recovery effect of the latter was better than that of the former, and the difference was statistically significant (P0.05, Table 1). 21-1 and Figure 21-1).

　　(2) Through statistical analysis of the gray-scale ratio of synaptophysin and internal reference protein in different groups, the expression of synaptophysin in the exercise training group and the ischemia control group was lower than that in the sham operation group, and the difference was statistically significant. The expression of synaptophysin after exercise training was higher than that of the model group (P\u003c0.01), which was statistically significant (P\u003c0.01). Conclusion: Treadmill training can improve the neuronal function scores of neurons in rats with cerebral ischemia, up-regulate the expression of synaptophysin, promote cerebral ischemia, and restore neurological function.