A method for screening AD rat models and evaluating recovery of AD rats after drug administration

Abstract

The present application belongs to the field of AD drug test using rats, and particularly relates to a method for screening AD rat model and a method for evaluating recovery of AD rats after taking drugs, the screening method comprising the following steps: conducting sugar water and hot water drinking test on known normal healthy rats and AD rats to be tested; if the AD rats to be tested significantly decrease water drinking times in S area compared with normal healthy rats, and significantly increase water drinking times in C area, the AD rats to be tested are AD rats, the method for evaluating recovery comprises the following steps: conducting sugar water and hot water drinking test on AD rats continuously taking drugs for several days, if the AD rats gradually increase water drinking times in S area with increasing days, and gradually decrease water drinking times in C area, it is indicated that the AD rats recover after taking drugs, the method of the present application can simply, quickly and painlessly screen qualified AD rats, and can evaluate recovery of AD rats after taking drugs by using a low-cost method, thereby effectively reducing development cost and time of AD specific drugs.

Description

Technical Field

[0001] This invention belongs to the field of AD drug testing using rats, specifically involving a method for screening AD rat models and evaluating the recovery of AD rats after drug administration. Background Technology

[0002] The evaluation of AD mice includes the identification of AD mice and the assessment of their recovery level. The identification of AD mice is of great significance for the selection of AD models and their behavioral performance, and helps to further understand the pathogenesis of AD; while the assessment of their recovery level is helpful in reflecting the curative effect of drugs and plays an important role in the development of new AD drugs with better efficacy.

[0003] Currently, common evaluation systems for AD rat models include the Morris water maze, MRI technology, and the detection of pathological biomarkers. The Morris water maze is used to study the impairment of learning and memory abilities in experimental animals; MRI technology is used to determine the lesion sites and pathogenesis of AD; and cellular and molecular level detection, due to its high specificity and wide variety, is often used for research on new drug development targets and early disease screening, and is currently a research focus. However, these methods have disadvantages such as being highly invasive to rats, requiring a high level of expertise from the experimenters, and being costly.

[0004] While both the Morris water maze and the Morris water maze are used to evaluate AD recovery in mice, the Morris water maze has higher standards and generates more experimental data, thus requiring stronger data processing skills from the experimenters. It also places higher demands on laboratory conditions, generally requiring consistent environmental parameters including temperature, sound, and even time. Furthermore, it necessitates experimental monitoring systems. Additionally, the species, physical condition, and even the mood of the experimental animals can affect the mice's swimming ability, thus influencing experimental results and potentially leading to significant errors. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for screening AD rat models to achieve efficient, convenient, low-cost, and non-invasive differentiation between AD rats and normal rats.

[0006] The technical solutions for achieving the above objectives include the following:

[0007] A method for screening AD rat models includes the following steps: Step 1, recording and saving the ultrasound emitted by physically strong rats when they drink sugar water;

[0008] Step 2: Set up a sugar water zone S and a spicy water zone C on both sides of the test field, and play the ultrasonic waves on one side of zone S;

[0009] Step 3: Establish standard values ​​using known normal healthy mice. Place normal healthy mice in the middle of the test area. Record the number of times the normal healthy mice drink water in the two areas within the specified time T1. To eliminate random errors, several repeated experiments should be conducted to obtain reliable data on the number of times normal healthy mice drink water in the two areas.

[0010] Step 4: Place the AD rats to be tested in the middle of the test area, and record the number of times the AD rats to be tested drink water in the two areas within the specified time T1.

[0011] Step 5: Compare the data recorded in Step 4 with those recorded in Step 3. If the number of times the rat drinks water in the S area is significantly lower in Step 4 than in Step 3, and the number of times the rat drinks water in the C area is significantly higher in Step 4 than in Step 3, then the AD rat to be tested is an AD rat.

[0012] Furthermore, the specified duration T1 is no less than 3 minutes.

[0013] Furthermore, in step 3, the repeated experiment is conducted no less than 8 times.

[0014] Rats are social rodents. Normal rats can transmit and receive ultrasound waves (USV) through specific structures in normal brain regions to alter their behavior and behavioral tendencies. Tests have shown that healthy rats emit positive sounds at 48-63 kHz when drinking sugar water. To ensure the effectiveness of these positive sounds, this invention records and stores the ultrasound waves of physically strong rats drinking sugar water. These physically strong rats are exceptionally healthy and strong mice selected through multiple physical and intelligence tests (such as endurance tests and water maze tests). When the ultrasound is played in the experimental field, if the brain regions of the AD rats to be tested are damaged, they will not be able to respond to the ultrasound, and their tendency to drink spicy water or sugar water will not show a significant bias (normal healthy rats will definitely have a significant preference). This allows us to screen out experimental subjects that meet the requirements for drug efficacy testing (identified as AD rats). Conversely, if the AD rats to be tested show a significant preference for drinking sugar water (the same behavior as normal healthy rats), it indicates that the brain regions of the AD rats to be tested can receive positive sounds and make correct (seeking benefits and avoiding harm) choices, proving that the AD rats to be tested are normal rats and are not suitable as experimental subjects for drug efficacy testing.

[0015] Another objective of this invention is to provide an easy-to-operate method for evaluating the recovery of AD mice after medication, with intuitive results.

[0016] The technical solutions for achieving the above objectives include the following:

[0017] A method for evaluating the recovery of AD mice after medication includes the following steps: Step 1, recording and saving the ultrasonic waves emitted by physically strong mice when they drink sugar water;

[0018] Step 2: Set up a sugar water zone S and a spicy water zone C on both sides of the test field, and play the ultrasonic waves on the S side;

[0019] Step 3: Administer the medication to AD mice continuously for several days. During the medication period, conduct several experiments. The experiments involve placing the medicated AD mice in the center of the experimental area and recording the number of times the medicated AD mice drink water in two designated areas within a specified time period T2.

[0020] 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 indicates that the AD mice are recovering after the medication (i.e. the medication is effective). The more obvious the upward or downward trend, the more effective the medication is.

[0021] Furthermore, the specified duration T2 shall not be less than 3 minutes.

[0022] Furthermore, the continuous medication period shall be no less than 14 days, and the number of trials shall be no less than 4.

[0023] The evaluation method for recovery after drug administration is consistent with the screening principle of the AD rat model mentioned above. The rats participating in the experiment are standard AD rats with brain damage. After drug administration, if the brain damage is repaired, their behavior will definitely be more like that of normal rats, thus being able to accept positive sounds and make correct (benefit-seeking and harm-avoidance) choices. In the experimental field, they will show a clear tendency to drink sugar water. As the drug administration time increases, the benefit-seeking and harm-avoidance choices will become more obvious. If each experiment is independent and undisturbed, and statistically significant data is obtained, it can prove that the drug has played a therapeutic role. If the rats put into the experimental field after drug administration are still unable to accept positive sounds or make correct choices, it indicates that the brain damage of the experimental rats remains unchanged, proving that the drug is ineffective.

[0024] The AD rat model screening method and the method for evaluating the recovery of AD rats after drug administration of the present invention have simple experimental requirements, can be carried out without professional technicians, are easy to operate, provide intuitive results, are low in cost, cause minimal trauma to animals, and improve the reliability of data. They have good application prospects.

[0025] It should be noted that the data on the number of times water was consumed in this case were obtained based on statistical analysis and several repeated experiments to eliminate the interference of accidental factors. In addition, the method of eliminating odor traces in the test site between two tests to prevent interference with the results of the subsequent test is a technical method commonly used in the field and will not be elaborated here. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the test field structure for an embodiment;

[0027] Figure 2 A comparison of the number of times normal healthy mice and AD mice drank sugar water and spicy water during the verification of the method for screening AD rat models in this embodiment;

[0028] Figure 3 The figure shows a comparison of the number of times three groups of rats drank sugar water and spicy water during the verification of the method for evaluating the recovery of AD rats after drug administration in the example. Detailed Implementation

[0029] The following examples demonstrate the specific verification of the two methods provided by the present invention.

[0030] See Figure 1 and Figure 2 The method for validating and screening AD rat models includes the following steps:

[0031] Record and save the ultrasonic waves emitted by physically dominant mice while they are drinking sugar water;

[0032] The experimental setup included a sugar water zone (S) and a spicy water zone (C). A professional loudspeaker played a USV (Unique Spoken Water) video on one side of the sugar water zone. Normal, healthy mice (N) were introduced from the center of the experimental area and allowed to explore freely for 3 minutes. The number of times the normal mice drank water in zones S and C was recorded. Afterward, the normal, healthy mice N were removed, and the cage containing their distinctive scent cues was promptly cleaned. The experiment was repeated using AD mice. Eight sets of experiments were repeated using non-repeated normal, healthy mice N and AD mice. The average number of times the N and AD mice drank spicy water and the average number of times they drank sugar water were calculated and compared in each set. See [link to relevant documentation]. Figure 2 The left side of the figure compares the number of times normal rats (N) and AD rats drank sugar water, while the right side compares the number of times normal rats (N) and AD rats drank spicy water. As can be seen from the figure, under the influence of ultrasound USV, AD rats drank sugar water significantly less often than normal healthy rats (N), while drinking spicy water significantly more often, which proves the effectiveness of the method for screening AD rat models of the present invention.

[0033] This invention provides a method for evaluating the recovery of AD mice after drug administration, see [link to relevant documentation]. Figure 1 and Figure 3 The following steps were used for verification, which were also conducted in... Figure 1 In the experimental setting shown, eight AD mice were randomly selected as the drug treatment group (using methimazole as an example). All mice in the drug treatment group received an intraperitoneal injection of 10 mg / kg. －1 ·d －1Memantine hydrochloride was administered for 14 consecutive days. Eight AD rats were randomly selected as the saline group, which received a sham injection of physiological saline. Eight healthy rats were randomly selected as the control group. The rats in each of the three groups were placed between the S and C zones, respectively, and allowed to explore freely for 3 minutes. The number of times the rats drank water in the S and C zones was recorded. The experiment was repeated to obtain statistically significant data on the number of times rats drank water in the S and C zones for each group. The same experiment was performed on days 1, 3, 7, and 14 after administration. Figure 3 The figure shows the water consumption of three groups of rats over 14 days. The left vertical axis represents the number of times they drank sugar water, and the right vertical axis represents the number of times they drank spicy water. Both the horizontal and vertical axes represent the number of days of drug administration. As can be seen from the figure, under the influence of ultrasound, the AD rats in the drug-treated group showed increasingly similar frequencies of sugar water and spicy water consumption to the normal group as the number of days of drug administration increased, indicating that the AD rats were gradually recovering (behaving more and more like normal healthy rats). In contrast, the saline group showed virtually no change in the frequency of sugar and spicy water consumption, proving that the method of evaluating the recovery of AD rats after drug administration can be used to assess drug efficacy.

[0034] This invention establishes a novel system for screening and evaluating the recovery of Alzheimer's disease (AD) rats based on the differences in how AD rats transmit and receive information via ultrasound compared to normal rats. This invention is minimally invasive to experimental animals, low-cost, easy to operate, and requires less sophisticated experimental environments and skilled personnel. It is highly reproducible and can serve as an effective evaluation system for AD rat recovery. It addresses the problems of high invasiveness, high skill requirements, and high costs associated with common AD rat model evaluation systems. This invention improves the evaluation process for AD rat model recovery after drug administration, significantly reducing research costs and increasing drug development efficiency.

Claims

1. A method for screening AD rat models, characterized in that, Includes the following steps: Step 1: Record and save the ultrasonic waves emitted by the physically strong rats when they drink sugar water; Step 2: Set up a sugar water zone S and a spicy water zone C on both sides of the test field, and play the ultrasonic waves on one side of zone S; Step 3: Establish standard values ​​using known normal healthy mice. Place normal healthy mice in the middle of the test area. Record the number of times the normal healthy mice drink water in the two areas within the specified time T1. To eliminate random errors, several repeated experiments should be conducted to obtain reliable data on the number of times normal healthy mice drink water in the two areas. Step 4: Place the AD rats to be tested in the middle of the test area, and record the number of times the AD rats to be tested drink water in the two areas within the specified time T1. Step 5: Compare the data recorded in Step 4 with those recorded in Step 3. If the number of times the rat drinks water in the S area is significantly lower in Step 4 than in Step 3, and the number of times the rat drinks water in the C area is significantly higher in Step 4 than 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 specified duration T1 shall not be less than 3 minutes.

3. The method for screening AD rat models according to claim 1, characterized in that, In step 3, the repeated experiment shall be conducted no less than 8 times.

4. A method for evaluating the recovery of AD mice after drug administration, characterized in that, Includes the following steps, Step 1: Record and save the ultrasonic waves emitted by the physically strong rats when they drink sugar water; Step 2: Set up a sugar water zone S and a spicy water zone C on both sides of the test field, and play the ultrasonic waves on the S side; Step 3: Administer the medication to AD mice continuously for several days. During the medication period, conduct several experiments. The experiments involve placing the medicated AD mice in the center of the experimental area and recording the number of times the medicated AD mice drink water in two designated areas within a specified time period T2. 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 indicates that the AD mice are recovering after medication. The more obvious the upward or downward trend, the more effective the medication is.

5. The method for evaluating the recovery of AD mice after drug administration according to claim 4, characterized in that, The specified duration T2 shall not be less than 3 minutes.

6. The method for evaluating the recovery of AD mice after drug administration according to claim 4, characterized in that, The duration of continuous medication shall be no less than 14 days, and the number of trials shall be no less than 4.

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