Method for screening AD rat model and evaluating recovery condition of AD rat after medication

Abstract

The invention belongs to the field of AD (Alzheimer's disease) drug tests by rats, and particularly relates to a method for screening AD rat models and a method for evaluating recovery conditions of AD rats after medication, and the screening method comprises the following steps: carrying out sweet water and spicy water drinking tests on known normal healthy rats and AD rats to be tested; if the water drinking times of the AD rats to be detected in the S area are obviously reduced relative to normal healthy rats and the water drinking times of the AD rats to be detected in the C area are obviously increased, the AD rats to be detected are AD rats, and the method for evaluating the recovery condition comprises the following steps: carrying out a sweet water and spicy water drinking test on the AD rats continuously taking medicines for several days, and if the water drinking times of the AD rats in the S area are gradually increased along with the increase of the date, judging that the AD rats to be detected are AD rats. If the water drinking frequency in the area C is gradually reduced, it is indicated that the AD rats recover after medication, the method can simply, rapidly and painlessly screen out the qualified AD rats, the recovery condition of the AD rats after medication can be evaluated by adopting a low-cost method, and the research and development cost and time of AD specific medicine are effectively reduced.

Description

Technical Field

[0001] The present invention belongs to the field of AD drug testing using rats, and specifically relates to a method for screening AD rat models and evaluating the recovery of AD rats after medication. Background Art

[0002] The evaluation of AD mice includes the identification of AD mice and the evaluation of their recovery degree. The identification of AD mice is of great significance for the selection of AD models and behavioral manifestations, which helps to further understand the pathogenic mechanism of AD; while the evaluation of their recovery degree is conducive to reflecting the curative effect of drugs and plays an important role in the research and development of new AD specific drugs with better effects.

[0003] At present, the common evaluation systems for AD rat models include Morris water maze, MRI technology, and detection of the expression of pathological markers. Morris water maze is used to study the impairment of learning and memory ability of experimental animals; MRI technology is used to determine the location of AD lesions and its pathogenesis; and the detection of cellular molecular levels is often used in the study of new drug development targets and early disease screening due to its high specificity and variety, and is the focus of current research. However, they have the disadvantages of being highly traumatic to rats, requiring high levels of experimenters, and being expensive.

[0004] The Morris water maze system is also used to evaluate the recovery of AD mice. The Morris water maze has a relatively high standard and a large amount of experimental data, so it requires a high level of data processing ability on the part of the experimenter. The laboratory conditions are also relatively high. The experimental environment is generally required to maintain consistency in temperature, sound, and even time. An experimental tracking and monitoring system is required. At the same time, the type, physique, and even mood of the experimental animals may affect the swimming ability of the mice and affect the experimental results, resulting in large errors. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for screening AD rat models so as to distinguish AD mice from normal mice efficiently, conveniently, at low cost and without damage.

[0006] The technical solution for achieving the above-mentioned purpose includes the following contents.

[0007] A method for screening an AD rat model comprises the following steps: step 1, recording and saving the ultrasonic waves emitted by the physically strong rats when drinking sugar water; Step 2, setting a sugar water area S and a spicy water area C on both sides of the test field, and playing the ultrasonic wave on one side of the S area; Step 3, use known normal healthy mice to establish standard values, put normal healthy mice in the middle of the test field, and record the number of times the normal healthy mice drink water in the two areas within the specified time T1. In order to eliminate accidental errors, several repeated tests should be carried out to obtain reliable numbers of times the normal healthy mice drink water in the two areas; Step 4, placing the AD rats to be tested in the middle of the test field, and recording the number of times the AD rats to be tested drink water in the two areas within a specified time period T1; Step 5, compare the data recorded in step 4 with the data recorded in step 3. If the drinking frequency of area S in step 4 is significantly reduced compared with that in step 3, and the drinking frequency of area C in step 4 is significantly increased compared with that in step 3, then the AD rat to be tested is an AD rat.

[0008] Furthermore, the prescribed duration T1 is not less than 3 minutes.

[0009] Furthermore, in step 3, the test is repeated no less than 8 times.

[0010] Rats are social rodents. Normal rats can transmit and receive ultrasound (USV) through specific structures in normal brain areas to transmit information and change their own behavior and action tendencies. After testing, normal healthy rats emit 48-63KHZ to express positive sounds when drinking sugar water. In order to ensure the effectiveness of positive sounds, the present invention records and saves the ultrasound of physically strong rats drinking sugar water. The physically strong rats are particularly healthy and strong rats selected through multiple physical and intelligence tests (such as endurance tests, water maze system tests, etc.). When the ultrasound is played in the experimental field, if the AD rat to be tested has damaged brain areas and cannot respond to the ultrasound, it will not have a clear preference for drinking spicy water or sugar water (normal healthy rats will inevitably have obvious preferences), thereby screening out experimental subjects that meet the requirements of drug efficacy testing (determined to be AD rats). On the contrary, if the AD rat to be tested has a clear preference for drinking sugar water (the same performance as normal healthy rats), it means that the brain area of ​​the AD rat to be tested can accept positive sounds and make the correct (seek benefits and avoid harm) choice, proving that the AD rat to be tested is a normal rat and is not suitable as an experimental subject for drug efficacy testing.

[0011] Another object of the present invention is to provide a method for evaluating the recovery of AD mice after medication, which is easy to operate and has intuitive results.

[0012] The technical solution to achieve the above-mentioned purpose includes the following contents.

[0013] A method for evaluating the recovery of AD mice after medication, comprising the following steps: Step 1, recording and saving the ultrasonic waves emitted by the physically strong mice when drinking sugar water; Step 2, setting a sugar water area S and a spicy water area C on both sides of the test field, and playing the ultrasonic wave on the S side; Step 3, the AD mice are continuously treated with the drug for several days. During the treatment period, several tests are carried out successively. The test is to put the AD mice treated with the drug in the middle of the test field, and within the specified time T2, the number of times the AD mice drink water in the two areas is recorded respectively. Step 4, compare the number of times the AD mice drank water in the two areas recorded in step 3. If the number of times the AD mice drank water in area S gradually increased with the date, and the number of times they drank water in area C gradually decreased, it means that the AD mice are recovering after medication (i.e., the medication is effective). The more obvious the upward or downward trend is, the more effective the medication is.

[0014] Furthermore, the prescribed duration T2 is not less than 3 minutes.

[0015] Furthermore, the continuous medication for a number of days is no less than 14 days, and the number of tests is no less than 4 times.

[0016] The method for evaluating recovery after medication is consistent with the principle of screening the above-mentioned AD rat model. The rats participating in the experiment are standard AD rats with brain damage. After medication, if the brain damage is repaired, their behavior will definitely be more like normal rats, so that they can accept positive sounds and make correct (seek benefits and avoid harm) choices. In the experimental field, they will show a clear tendency to drink sugar water. As the medication time increases, the choice of seeking benefits and avoiding harm will become more obvious. If each experiment is independent and undisturbed, obtaining statistically significant data can prove that the drug has exerted its therapeutic function; if the rats put into the experimental field after medication are still unable to receive positive sounds or make correct choices, it means that the brain damage of the experimental rats remains the same, proving that the drug is ineffective.

[0017] The AD rat model screening method and the method for evaluating the recovery of AD rats after medication of the present invention require simple experimental conditions, can be performed without professional technicians, are easy to operate, have intuitive results, are low in cost, cause little trauma to animals, and improve the reliability of data. They have good application prospects.

[0018] It should be noted that the recorded data on the number of times of drinking water described in this case were obtained by conducting several repeated tests on a statistical basis to eliminate the interference of accidental factors. In addition, between the two tests, the means of eliminating odor traces in the test field to prevent interference with the results of the next test are commonly used technical means in this field and will not be elaborated in this case. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A schematic diagram of the test field structure of an embodiment; Figure 2 This is a comparison chart of the number of times normal healthy mice and AD mice drank sugar water and spicy water when validating the method for screening AD rat models in the embodiment; Figure 3 This is a comparison chart of the number of times the three groups of rats drank sugar water and spicy water when validating the method for evaluating the recovery of AD mice after medication in the embodiment. DETAILED DESCRIPTION

[0020] The two methods provided by the present invention are specifically verified in combination with the embodiments below.

[0021] See also Figure 1 and Figure 2 , verifying the method for screening AD rat models, comprising the following steps, Record and save the ultrasonic USVs emitted by the physically strong mice when they drink sugar water; The test field is set up with a sugar water area (S) and a spicy water area (C). A professional broadcaster is used to play USV on one side of the sugar water area. A normal healthy mouse (N) is placed in the middle of the test field and allowed to explore freely for 3 minutes. The number of times the normal mouse drinks water in S and C is recorded. Then the normal healthy mouse N is taken out and the unique odor clues in the cage are cleaned in time. Repeat the experiment with AD mice. Repeat 8 groups with non-repeated normal healthy mice N and AD mice, and calculate the average number of spicy and sugar drinking times of N and AD mice in each group and compare them. See Figure 2 The left part of the figure shows the comparison of the number of times normal mice N and AD mice drank sugar water, and the right part of the figure shows the comparison of the number of times normal mice N and AD mice drank spicy water. It can be seen from the figure that under the influence of ultrasonic USV, the number of times AD mice drank sugar water was significantly reduced compared with normal healthy mice N, while the number of times they drank spicy water was significantly increased, which proves the effectiveness of the method for screening AD rat models of the present invention.

[0022] The present invention provides a method for evaluating the recovery of AD mice after medication, see Figure 1 and Figure 3 , the following steps were used for verification, also in Figure 1 In the test field shown in the figure, eight AD mice were randomly divided into the drug-administered group (here, memantine was used as an example). The drug-administered group was intraperitoneally injected with 10 mg kg －1 ·d －1 Memantine hydrochloride was administered for 14 consecutive days. Eight AD rats were randomly selected as the saline group, and the saline group was injected with saline by sham surgery. Eight healthy rats were randomly selected as the normal group. The rats in the three groups were placed between the S area and the C area respectively, and allowed to explore freely for 3 minutes. The number of times the rats drank water in S and C was recorded. After repeated experiments, the number of times each group of rats drank water in S and C with statistical significance was obtained. The same experiment was performed on the 1st, 3rd, 7th, and 14th days of administration. Figure 3The figure shows the drinking water situation of three groups of rats within 14 days. The left vertical axis in the figure represents the number of times of drinking sugar water, the right vertical axis represents the number of times of drinking spicy water, and the horizontal and vertical axes both represent the number of days of medication. As can be seen from the figure, under the influence of ultrasound, the AD rats in the medication group are getting closer and closer to the normal group as the number of days of medication increases, whether they drink sugar water or spicy water, indicating that the AD rats are gradually recovering (behaving more and more like normal healthy rats), while the salt water group has basically no change in the number of times of drinking sugar and spicy water, proving that the method of evaluating the recovery of AD rats after medication of the present invention can be used to evaluate the efficacy of the drug.

[0023] The present invention is based on the difference between AD mice and normal mice in transmitting and receiving information using ultrasound, and completes the establishment of a new system for screening and evaluating the recovery of AD mice. The present invention has low trauma to experimental animals, low cost, convenient operation, low requirements on the experimental environment and the professional ability of the experimenter, and high repeatability. It can be used as an effective evaluation system for the recovery of AD mice, solving the problems of the common evaluation system of AD rat models, such as high trauma to rats, high requirements on the level of experimenters, and high cost. It improves the evaluation process of the recovery of AD rat models after medication in the future AD new drug development research and drug dosage form selection, greatly reducing research costs and improving drug development efficiency.

Claims

1. A method for screening AD rat models, characterized in that: The steps include: Step 1, record and save the ultrasonic waves emitted by the strong mice when drinking sugar water; Step 2, setting a sugar water area S and a spicy water area C on both sides of the test field, and playing the ultrasonic wave on one side of the S area; Step 3, use known normal healthy mice to establish standard values, put normal healthy mice in the middle of the test field, and record the number of times the normal healthy mice drink water in the two areas within the specified time T1. In order to eliminate accidental errors, several repeated tests should be carried out to obtain reliable numbers of times the normal healthy mice drink water in the two areas; Step 4, placing the AD rats to be tested in the middle of the test field, and recording the number of times the AD rats to be tested drink water in the two areas within a specified time period T1; Step 5, compare the data recorded in step 4 with the data recorded in step 3. If the drinking frequency of area S in step 4 is significantly reduced compared with that in step 3, and the drinking frequency of area C in step 4 is significantly increased compared with that in step 3, then the AD rat to be tested is an AD rat.

2. The method for screening AD rat models according to claim 1, characterized in that: The prescribed duration T1 is no less than 3 minutes.

3. The method for screening AD rat models according to claim 1, characterized in that: In step 3, the test is repeated no less than 8 times.

4. A method for evaluating the recovery of AD mice after medication, characterized in that: The following steps are included: Step 1, record and save the ultrasonic waves emitted by the strong mice when drinking sugar water; Step 2, setting a sugar water area S and a spicy water area C on both sides of the test field, and playing the ultrasonic wave on the S side; Step 3, the AD mice are continuously treated with the drug for several days. During the treatment period, several tests are carried out successively. The test is to put the AD mice treated with the drug in the middle of the test field, and within the specified time T2, the number of times the AD mice drink water in the two areas is recorded respectively. Step 4, compare the number of times the AD mice drank water in the two areas recorded in step 3. If the number of times the AD mice drank water in area S gradually increased with the date, and the number of times they drank water in area C gradually decreased, it means that the AD mice are recovering after medication. The more obvious the upward or downward trend is, the more effective the medication is.

5. The method for evaluating the recovery of AD mice after medication according to claim 4, characterized in that: The prescribed duration T2 is no less than 3 minutes.

6. The method for evaluating the recovery of AD mice after medication according to claim 4, characterized in that: The continuous medication for a number of days is no less than 14 days, and the number of tests is no less than 4 times.

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