Experimental study on the animal model of dementia induced by injection of 1922 IgG2 saporin into the lateral ventricle

Summary

Rats with senile dementia were established and their learning and memory abilities and the number of basal forebrain cholinergic neurons were observed. In the left lateral ventricle of adult SD rats, the immunotoxin 1922 IgG2 saporin (2.5 μg/5 μl) was injected. After 3 weeks, the learning and memory ability was detected by Y maze. After 4 weeks, the rats were sacrificed and the groups were observed by immunohistochemistry combined with image analysis technique. Changes in the number of ChAT-positive neurons in the basal forebrain of rats. The results showed that the learning and memory ability of the model group was significantly lower than that of the normal group (P<0.01). The medial septal nucleus (MS) and the vertical portion (VDB) of the basal forebrain of the model group were cholinergic. The number of neurons decreased to 9.70% and 14.09% of the normal group, respectively, which was significantly lower than that of the normal group (P<0.01). The learning and memory ability were closely related to the number of basal forebrain cholinergic neurons. The above results indicate that the animal model of AD rat can be successfully established by using 1922 IgG2 saporin immunotoxin, which can be used for the screening of anti-dementia drugs and the evaluation of drug efficacy. The main symptoms of Alzheimer's disease (AD) are progressive cognitive dysfunction and impaired learning and memory, and their incidence increases with age. So far, due to the complexity of the pathogenesis of AD, there is a lack of effective treatment measures in the clinic. Establishing a reliable animal model of AD is an important part of studying AD. In recent years, domestic and foreign scholars have established some AD models to explore the pathogenesis of AD and develop effective therapeutic drugs. However, there is no ideal model to reproduce this. All the characteristics of the disease. The degeneration of the acetylcholine (ACh) energy system in the brain is considered to be one of the main pathological factors of AD. In this experiment, a selective immunotoxin 1922 IgG2 saporin [124] was injected into the lateral ventricle of adult SD rats to establish AD. Animal models were observed for learning and memory and changes in basal forebrain cholinergic neurons.

Materials and Methods

Material

The acetylcholine transferase (ChAT) monoclonal antibody was purchased from Chemicon, and the Ultrasensitive SP kit and DAB chromogenic kit were all manufactured by Maxin Biotech Inc. Twenty-four adult male Sprague2 Dawley (SD) rats weighing 250-300 g were randomly divided into: (1) normal group (n=8); (2) saline group (n=8): saline injected into the left lateral ventricle 5 μg / 5 μl of the solution of the 192 IgG2sa2porin solution was injected into the left lateral ventricle (5).

2. Animal model making

The rats in the model group were intraperitoneally injected with 1% sodium pentobarbital (40 mg/kg), and the animals were anesthetized and fixed on the stereotaxic instrument with a flat head position. All instruments were soaked overnight with 1% Xinjie and the solution (add 1% sodium nitrite to prevent rust). Disinfect with 3% iodine and 75% alcohol. The epidermis was cut in the median sagittal position along the cranial ridge, the periosteum was scraped off, and cauterized with 3% H2O2. Make the skull white, use a sterile cotton swab to fully open the wound, refer to the stereotactic map of the rat, before the 囟 囟 zero point, press the front 囟 0. 8mm, the outer 1. 5mm coordinates with a 7-gauge needle drill a hole, with 10μl The micro-injector took 1922 IgG2 saporin 2. 5 μg / 5 μl, the needle was inserted vertically from the hole, and the needle was deep to the ventral side 5. 5 mm, slowly and constantly, the time was not less than 5 min, and the needle was left for 3 min. The wound is disinfected and the skin is sutured. In the saline group, 5 μl of physiological saline was injected in the same manner. After 3 weeks of feeding, the Y-maze behavior test was performed. The rats were sacrificed 4 weeks later. The changes of the number of ChAT-positive neurons in the basal forebrain of each group were observed by immunohistochemistry combined with image analysis.

3. Y maze behavior detection

After 3 weeks, each group of rats was tested in a three-equivalent radial labyrinth box. The top of each arm of the box was equipped with a signal light, the light area was a safe area, and the orientation of the safety area was randomly changed. The voltage is 70V and the electric shock is delayed for 30s. Train the rats to learn to distinguish the safety lights and actively escape the electric shock. After the conditional avoidance reaction was established, the rats were given 10 tests every day and afternoon, and the training was continued for 6 days. Among them, 9 of the 10 tests were correct as the standard. Record the number of training required for each rat to reach the standard of learning as the learning ability (the fewer the number, the stronger the learning ability, and the weaker the other); the next 24 hours after reaching the standard of the society, the correct number of times is the memory ability (more times) , indicating that memory is strong, and vice versa).

4. Tissue sectioning and dyeing

Anesthetized animals were rapidly perfused with 200 ml of normal saline through the left ventricle 2 liters of aorta until the liver color became white and then reperfused with 4% paraformaldehyde solution 250 ml (0.1 mol/LPB, pH 7.4). The principle is completed within 60 minutes. The brain was quickly opened and placed in the same fixing solution at 4 ° C for about 2 to 4 hours. Then the brain tissue was transferred to a 15% sucrose solution (0.1 mol/LPB, pH 7.4) and placed in a refrigerator at 4 ° C overnight. , refilled with 30% sucrose solution (0.1 mol/LPB formulation, pH 7.4) at 4 ° C overnight. The brain tissue was coronally serially sliced ​​with a cryostat slicer, and the slice thickness was 40 μm, and the sections were collected with PBS. The section begins at the initial level of the corpus callosum and ends at the leading edge of the anterior commissure. Two slices are taken every 3 slices and immunohistochemical staining is performed according to the ABC method. The sections were first washed with 0. 01mol/L PBS (pH 7.2) for 5 min×3 times; each section was treated with a drop of peroxidase blocker for 30 min, PBS washed for 5 min×3 times; and then one drop of normal non-immune animals was added. Serum was treated for 30 min; after blotting the reagents, 50 μl of ChAT monoclonal antibody solution (1:800) was added to each section and incubated overnight at 4 °C in a refrigerator. This day, wash with PBS for 5 min × 3 times; according to the above method, biotin-labeled secondary antibody and streptomycin antibiotic protein-peroxidase were added in order, and finally freshly prepared DAB coloring solution (850 ml double) was added to each section. Steamed water plus DAB kit A, B, C each 1 drop) 50μl color for 3 ~ 6min, after observation under the microscope, the color is satisfactory, rinse with PBS, patch, dry and dry, gradient alcohol dehydration, xylene transparent, neutral gum Covering.

5. Computer acquisition and counting

Each rat selected 5 representative planes of the 2 oblique angle complex, and each plane selects the inner medial nucleus under the low power microscope (4 ×)

(MS) and the oblique section of the oblique section (VDB) to observe the field of view, and then the MS and VDB cell dense areas in 2 fields of view under the mid-magnification (20×), using a fully automatic image analysis system (Germany KONTRONIBAS2.0, JVCky2F30B32CCD) The color image recording input device displays the visual field, and quantitatively detects the number, area and circumference of the positive neurons through image analysis processing; and observes the neuron morphology under high magnification (40×).

6. Statistical methods

All experimental data are measurement data and are expressed as mean ± standard deviation (x ± s). Statistical analysis was performed using SPSS 11.0 statistical software. The t-test was used to compare the data of each group. The correlation between the two variables was analyzed by linear correlation regression.

Result

1. Comparison of learning and memory ability of rats in each group Y-maze test results of learning and memory ability of rats in each group are shown in the table. Paired t-test showed that the learning and memory ability of the model group rats decreased significantly, and the normal group and physiology The salt water group is more obvious

Sexual differences (P < 0.01).