动物造模,混合性记忆障碍模型 z-bio 2023-01-18 505

　　(1) Establishing a model of learning and memory impairment by repeated cerebral ischemia and reperfusion

　　1. Modeling materials Animal: Kunming mouse, male, 25-28g; Medicine: chloral hydrate; Apparatus: constant temperature electric heating box.

　　2. Modeling method After anesthesia with chloral hydrate, the bilateral common carotid arteries were separated by a median incision in the lower neck, and the arteries were clamped with non-invasive arterial clamps. The method of ischemia was 15 minutes of ischemia, 15 minutes of reperfusion, and 15 minutes of re-ischemia. And at the first ischemia, the rat's tail was cut and exsanguinated at 1cm, resulting in hypotension. In the sham operation group, the arteries were not clamped and blood was not bleeding. During the ischemia process, a constant temperature electric heating box was used to keep the mouse body temperature at (36.2±1.2)℃. Training and testing the results of water labyrinth 4 days after surgery.

　　3. Principles of Modeling A model of learning and memory impairment caused by repeated cerebral ischemia and reperfusion in mice was used to establish a model.

　　4. The swimming time of mice in the water labyrinth experiment generally changes after the model is made. The time for the model mice is (168.3±75.1) min, which is significantly longer than (24.7±15.5) min in the sham operation group; Model mice were made (17.9±11.1) times, significantly more than the sham operation group (2.5±1.6) times. It shows that the model is successful.

　　5. Biochemical changes after modeling. The level of lactic acid in the brain tissue of mice was (28.06±0.79) mmol/g in the model mice and (24.40±2.02) mmol/g in the sham operation group. There was a significant difference.

　　6. Matters needing attention Strictly sterilize surgical instruments to prevent surgical infection, surgical trauma should be as small as possible, and strictly aseptic operation. Keep the animal's optimum temperature and humidity as much as possible in the breeding room.

　　(2) Temporary cerebral ischemia method to establish learning and memory impairment model

　　1. Modeling materials Animal: Kunming mouse, male, 18-22g; Drug: anesthetic.

　　2. Modeling method After the animal is anesthetized, fix it in the supine position. Cut the skin about 7mm in the middle of the neck, separate the bilateral common carotid arteries, place a white silk thread 0 under the left artery, and black silk thread 0 under the right artery to block blood flow. The two ends of each line are aligned, and the 4 terminals are passed through a section of PE50 plastic pipe with a length of about 5mm. The skin is penetrated from the middle of the back of the neck, and the plastic tube is buried in the skin. Use another black silk thread to pass through the black and white thread to cover the loop of the blood vessel. During ischemia, use a pair of tweezers to gently press the skin on the back of the neck, tighten the ends of the 4 black and white wires, and fix the 4 wires with the skin with arterial clips, resulting in cerebral ischemia. After the ischemia is over, loosen the arterial clamp and gently pull the lower neck lead to completely restore the blood reperfusion of the common carotid artery.

　　3. Principles of Modeling A model of learning and memory impairment caused by transient cerebral ischemia in mice is used to establish a model.

　　4. Changes after modeling After the bilateral common carotid arteries are completely blocked, convulsions first appear, body temperature decreases, breathing slows down, and finally the righting reflex disappears. Animals may die during ischemia. After the blood perfusion was restored, the animal's righting reflex gradually recovered, breathing accelerated, and basically returned to normal activities within 3 to 5 hours. The body temperature of animals in the sham operation group did not change significantly.

　　The platform jumping experiment showed that compared with the animals in the sham operation group, the number of errors in learning in the sham operation group was (1.77±0.73) times, the number of errors in memory was (0.55±0.93) times, and the escape latency during learning , EL) is (23.8±31.1)s, the step-down latency (SDL) during memory is (282.7±36.4)s; the number of errors during module learning is (3.00±2.16), and the number of errors during memory is (1.58±1.16) times, the incubation period is (24.8±31.0) s, and the memory is (140.4±120.3) s. Poor academic performance is manifested by prolonged reaction time and increased number of errors.

　　The dark avoidance experiment showed that compared with the animals in the sham operation group, the sham operation group had a learning latency of (157.0±34.3)s, a memory latency of (297.6±19.6)s, and the number of errors during learning was (2.27± 1.62) times, the number of errors during memory is (0.27±0.65) times; the latency of module learning is (157.4±24.6)s, the latency of memory is (187.8±72.4)s, and the number of errors during learning is (1.83±1.11) ) Times, the number of errors in memory is (3.00±1.33) times.

　　5. Precautions Strictly sterilize surgical instruments to prevent surgical infection, surgical trauma should be as small as possible, and strictly aseptic operation. Keep the animal's optimum temperature and humidity as much as possible in the breeding room.

　　(3) Rat model of memory impairment with permanent ligation of bilateral common carotid arteries

　　1. Modeling materials Animal: Wistar rat, male, 280～300g; Medicine: Pentobarbital (40mg/kg).

　　2. Modeling method After the animal is anesthetized, fix it in the supine position. After sterilizing the middle of the neck, the incision was made, the bilateral common carotid arteries were separated and ligated with silk thread, and the incision was sutured. Animals in the sham operation group were treated the same as those in the ligation group except that bilateral common carotid arteries were not ligated. The two groups of animals were raised under the same conditions.

　　3. Principles of Modeling A model of learning and memory impairment caused by permanent cerebral ischemia in rats is used to establish a model.

　　4. Changes after modeling The mortality rate of animals within 24 hours after bilateral common carotid artery ligation is about 30%, and the mortality rate after 48 hours is zero. After ligation, most rats have drooping eyelids, low mobility and reduced spontaneous activity, but a few rats have increased spontaneous movement after surgery. All the surviving rats returned to normal on the second day after surgery. After 30 days of survival, obvious learning and memory impairment may appear; after 60 days, the learning and memory ability is still reduced, and there is no trend of recovery.

　　5. Biochemical and pathological changes after modeling 90h postoperatively, Nissl bodies in the hippocampal pyramidal cell cytoplasm were reduced or defective, and apoptotic bodies appeared. After 60 days, the density of hippocampal pyramidal cells decreased significantly.

　　6. Matters needing attention Strictly sterilize surgical instruments to prevent surgical infection, surgical trauma should be as small as possible, and strictly aseptic operation. Keep the animal's optimum temperature and humidity as much as possible in the breeding room.

　　(4) Cerebral ischemia-reperfusion-induced learning and memory impairment in mice

　　1. Modeling materials Animals: male KM mice, 28～33g; medicine: 4% chloral hydrate.

　　2. Modeling method The experimental mice were fasted for 12 hours before the operation, and they were free to drink water. After intraperitoneal injection of 4% chloral hydrate (0.1ml/10g body weight) anesthetized, the rats were fixed on the dissection board in the supine position, the middle of the neck was shaved, the skin was disinfected with iodine and 75% alcohol, and the neck was cut longitudinally along the middle line of the front and back direction. The skin is about 0.5cm, the tissues and muscles are bluntly separated, the bilateral common carotid arteries are exposed and separated, and a silk thread is threaded down for use. Be careful not to damage the vagus nerve. A 0.5cm incision was made above the clavicle to separate the common jugular vein. Intubation (pre-soaked in heparin) at the common jugular vein to draw blood to lower blood pressure (30% of the total blood volume). The bilateral common carotid arteries were clamped for 20 minutes with non-invasive microarterial clips. At the end of the timer, loosen the arterial clamp, return blood, pull out the catheter, and suture the wound. The animals were randomly grouped into: sham operation group and model group. The anesthesia and operation procedures of the sham operation group are the same as those of the model group, but the blood flow is not blocked and bloodletting is not performed to reduce errors caused by surgical damage. Spontaneous activity and passive avoidance experiments were carried out on the 15th day after surgery, and the spatially resolved learning and memory experiment was carried out on the 17th day.

　　3. Principle of Modeling Brain tissue is very sensitive to ischemia and hypoxia. A mouse model of learning and memory impairment was established by closing the bilateral common carotid artery clamps and taking blood to lower the blood pressure and then reinfusing it.

　　4. Compared with the sham operation group, the general changes after the model were made. The spontaneous activities of mice in the model group were significantly reduced. The sham operation group (149.2±34.6) times, the model group (102.4±31.2) times; the model group performed significantly in the platform learning and memory test The performance is worse than that of the sham operation group, which is represented by the training latency [sham operation group (6.45±6.35) s, model group (13.09±9.45) s] and electric shock time [sham operation group (15.55±10.27) s, model Group (25.91±11.16)s] increased significantly; the number of errors in memory scores [sham operation group (1.36±1.21) times, model group (3.91±2.43) times] increased significantly, and the incubation period of step down [sham operation group (217.1±65.6) )s, model group (18.3±21.0)s], total stay time on stage [sham operation group (239.5±45.7)s, model group (63.27±63.89)s] significantly decreased.

　　Compared with the sham operation group, the spatial resolution learning and memory performance of the model group was significantly reduced. On the third day of training, the latency of the model group seeking platform. [sham operation group (22.00±11.26)s, model group (83.91±63.02)s] significantly increased , Training on the 4th day of the platform seeking latency [sham operation group (14.91±5.47) s, model group (37.91±37.38) s], training on the 5th day of the platform seeking latency [sham operation group (12.91±1.58) s, model group ( 24.73±17.32)s] There is a significant difference between the model group and the sham operation group. The number of errors in the model group increased significantly [(3.82±1.89) times in the sham operation group on the 3rd day of training, (8.91±6.09) times in the model group; (2.55±1.13) times in the sham operation group on the 4th day of training, (4.27±2.45) in the model group ) Times; sham operation group (3.36±0.67) times, model group (3.64±1.57) times].

　　5. Biochemical and pathological changes after modeling. After staining with hematoxylin and eosin (HE), under light microscope, it can be seen that the number of hippocampal neurons in the sham-operated group was large, multi-layered, and neatly arranged. The nucleus is large and clear, with obvious nucleoli. The number of hippocampal CAI neurons in the model group was reduced, the loss was obvious, the levels were few and unclear, the nucleus was constricted into triangles or polygons, and the nucleus was densely stained.

　　Compared with the sham operation group, the total antioxidant capacity of mouse brain homogenate in the model group [sham operation group (0.48±0.04) U/mg·prot, model group (0.24±0.04) U/mg·prot], total superoxide The activity of superoxide dismutase (SOD) decreased significantly [sham operation group (123.5±14.5) U/mg·prot, model group (66.9±13.9) U/mg·prot], in the mouse brain homogenate of the model group The content of malondialdehyde (MDA) was significantly higher than that in the sham operation group [sham operation group (0.59±0.15) nmol/mg·prot, model group (1.17±0.24) nmoL/mg·prot], indicating that ischemia and reperfusion caused Lipid peroxidation damage.

　　6. Precautions Observe the animal's body temperature during all the modeling process, and keep the animal's anal temperature at about (36±0.5)℃ to prevent the protective effect of hypothermia on cerebral ischemia injury.

　　(5) Low-iron feed, zinc-deficient and high-zinc feed lead to learning and memory impairment model

　　1. Modeling materials Animals: healthy rats; drugs: low-iron feed (iron content of 11.9mg/kg), deionized water, zinc deficiency (<2mg/kg) and high zinc (1000mg/kg) feed.

　　2. Method of Modeling Rats were fed with low-iron feed (iron content of 11.9 mg/kg) and deionized water for 10 weeks. Or zinc deficiency (<2mg/kg) or high-zinc feed (1000mg/kg), deionized water feeding rats for 4 weeks.

　　3. Principles of Modeling Lack of iron, zinc, and excessive zinc and aluminum can cause learning and memory disorders.

　　4. General changes after model building. After 10 weeks of feeding on low-iron feed, the test showed memory impairment. Zinc-deficient and high-zinc feed were fed for 4 weeks and tested, and both showed memory impairment.

　　5. Biochemical and pathological changes after modeling. The iron content and monoamine oxidase activity in the brain tissue of rats fed with low-iron feed were significantly reduced, the content of norepinephrine and 5-hydroxytryptamine (5-hydroxytryptamine, 5-HT) in the cerebral cortex increased, and the nerves in the CA3 area The glial cell foot process is swollen and the organelles are sparse. In the zinc-deficient feed feeding group, serum and brain zinc and iron content decreased, and copper increased; in the high-zinc group, serum zinc was significantly increased, and serum and brain copper and iron content were significantly reduced. The brain tissue 5-HT of the two groups was significantly reduced, and 5-hydroxyl Indole acetic acid (5-hydroxyindoleacetic acid, 5-HIAA) was significantly increased.