Method for simultaneously constructing anxiety and memory impaired mouse models

Abstract

The invention discloses a method for simultaneously constructing anxiety and memory impaired mouse models, which comprises the following steps: (1) adaptive feeding: randomly performing adaptive feeding on two groups of mice under the same feeding conditions, the feeding environment temperature is 22-26 DEG C, the humidity is 40-60% RH, the illumination and the night are alternately performed for 12 hours, and the mice are fed for a week; (2) dividing the two groups of mice into a control group and an experimental group, feeding the control group in an environment with the temperature of 22-26 DEG C and the humidity of 40-60% RH, feeding the experimental group in an environment with the temperature of 33-36 DEG C and the humidity of 80-95% RH, alternately performing illumination and night for 12 hours, and continuously feeding for at least four weeks; and (3) respectively carrying out behavioral detection of an elevated cross maze, an open field experiment, a Y maze and a new object identification experiment on the mice in the control group and the experimental group after being fed in the step (2). According to the invention, external environmental stimulation is adopted, drug influence is avoided, no harm is caused to mice, and the device is more humanized.

Description

Technical Field

[0001] The invention relates to a method for simultaneously constructing an anxiety mouse model and a memory-impaired mouse model, and belongs to the technical field of medical evaluation and detection. Background Art

[0002] In mouse modeling, different diseases correspond to different mouse models, such as depression mouse model, autism mouse model, acute lymphoblastic leukemia mouse model, primary liver cancer mouse model, anxiety mouse model, etc. Drug injection and attack on mice are commonly used methods to establish these models.

[0003] The existing mouse models of mental illness are mainly established through drug injection. However, after drug injection, the expression of mice at the molecular level will also be affected. When we study the impact of other factors on mental illness, it will be affected by the drug, resulting in some changes at the molecular level that may not conform to the original trend of change. Therefore, it is necessary to find a new mouse modeling method that does not inject drugs into mice, so as to avoid the impact of drug injection and make the study of the impact of other factors on mental illness more accurate. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new method for constructing a mouse model, which does not require injection of drugs or external attack on mice, and can simultaneously construct an anxiety mouse model and a memory-impaired mouse model.

[0005] In order to solve the above technical problems, the present invention discloses a method for simultaneously constructing an anxiety and memory impairment mouse model, the method comprising the following steps:

[0006] (1) Adaptive feeding: The mice were randomly divided into two groups for adaptive feeding. The two groups were fed under the same feeding conditions, with a feeding environment temperature of 22-26°C, a humidity of 40-60% RH, and light and darkness alternating for 12 h each. The feeding was continued for one week.

[0007] (2) The two groups of mice after adaptive feeding in step (1) are divided into a control group and an experimental group. The control group is raised in an environment with a temperature of 22-26° C. and a humidity of 40-60% RH, and the experimental group is raised in an environment with a temperature of 33-36° C. and a humidity of 80-95% RH, with light and darkness alternating for 12 hours each, and the continuous raising time is at least four weeks;

[0008] (3) Behavioral testing of mice: The mice in the control group and the experimental group fed in step (2) were subjected to behavioral testing of elevated plus maze, open field test, Y maze and novel object recognition test, respectively.

[0009] Furthermore, the adaptive feeding in step (1) and the feeding environment of the control group in step (2) are both SPF-grade experimental animal rooms; the feeding environment of the experimental group in step (2) is an artificial climate box. SPF (specific pathogen free) grade experimental animals, that is, animals without specific pathogens, do not carry major potential infections or conditional pathogens and pathogens that interfere greatly with scientific experiments. SPF-grade experimental animal rooms are places that can provide experimental animals that meet this level in a barrier system or isolation system. SPF-grade experimental animal rooms implement strict microbial control, and the experimental environment strictly controls the entry and exit of personnel, objects, and air.

[0010] Furthermore, during the adaptive feeding in step (1) and the feeding of the control group and the experimental group in step (2), the mice were free to eat and drink, and the feed and drinking water used by the two groups of mice were kept consistent.

[0011] Furthermore, in step (3), the behavioral test of the elevated plus maze is specifically as follows: the mouse is placed in the center of the plus maze with its head facing the open arms, and timing is started after the mouse enters the maze. The monitoring instrument automatically monitors the number of times and time the mouse enters the open arms within 5 minutes.

[0012] Furthermore, in step (3), the behavioral detection of the open field experiment is specifically as follows: placing the mouse in the center of the open field, timing the mouse from the time it enters the open field, and the monitoring instrument automatically monitors the time the mouse spends in the center of the open field and the distance it moves in the open field within 5 minutes to 10 minutes.

[0013] Furthermore, in step (3), the Y-maze behavioral test is specifically as follows: the mouse is placed with its head facing the junction of the two arms of the Y-maze, timing is started from the moment the mouse enters the Y-maze, and the order in which the mouse enters each arm within 5 minutes to 10 minutes is monitored. If the mouse does not enter the same arm repeatedly for three consecutive times, it is recorded as correct, otherwise it is recorded as the opposite. Then the number of spontaneous alternations is recorded, and the spontaneous alternation rate is calculated based on the recorded number of spontaneous alternations. Spontaneous alternation rate (%) = [(number of spontaneous alternations) / (total number of arm entries-2)]×100%.

[0014] Further, in step (3), the behavioral detection of novel object recognition is specifically as follows: on the first day, the mice are habituated to the test room without objects for 5 min-10 min, and then returned to the original feeding environment; on the second day, the mice are returned to the test room, and in the presence of two original objects, a video is collected for 5 min-10 min, and then the mice are returned to the original feeding environment; on the third day, the mice are returned to the test room, and stay in the presence of an original object and a new object for 5 min-10 min, and the instrument automatically monitors the number and time of the mouse exploring the new object and the original object within the 5 min-10 min, and the time used for habituation to the test room on the first day, the time for collecting videos on the second day, and the monitored time of the mouse exploring the new object and the original object on the third day are consistent. The novel object discrimination index is calculated based on the monitored novel object exploration time and original object exploration time, novel object discrimination index (%) = novel object exploration time / (new object exploration time + original object exploration time) × 100%, and when the discrimination index is greater than 50%, it indicates that the animal pays more attention to the novel object.

[0015] Furthermore, the test chamber is at least 50×50×40 cm 3 A square space of 100×100×40m 3 In the square space, the shapes of the original object and the new object are obviously different.

[0016] Furthermore, the mice are 6-8 weeks old and in puberty.

[0017] Furthermore, the illumination time in step (1) is from 8:00 am to 8:00 pm, and the night time is from 8:00 pm to 8:00 am.

[0018] The present invention regulates the living temperature and humidity of mice, and after a certain period, i.e., continuous high temperature and high humidity stimulation, it is observed that the anxiety level and memory of mice have changed, and a mouse anxiety model and a mouse memory impairment model are successfully established. About more than 55% of the areas in my country belong to high temperature and high humidity environments, and the number of hot and humid weather days each year tends to increase year by year. People's production and life are affected by heat, physiological functions related to body temperature regulation are tense, and psychological states are unbalanced, which seriously threatens occupational health and personal safety. Therefore, the present invention has practical significance for constructing a mouse model through high temperature and high humidity stimulation, and provides an experimental basis for the later regulation of high temperature and humidity to bring adverse effects to people.

[0019] The method for constructing a mouse model provided by the present invention uses external environmental stimulation and does not inject drugs into the mice, thereby avoiding the influence of the injected drugs when studying the influence of other factors on mental illness, and avoiding the occurrence of changes at the molecular level that may not conform to the original change trend of mental illness. The present invention does not cause harm to the mice, and the mice are protected by humanitarianism, which is more humane. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the elevated plus maze behavioral test results of mice in Example 1 of the present invention;

[0021] Figure 2 This is a graph showing the open field behavior test results of mice in Example 1 of the present invention;

[0022] Figure 3 This is a diagram showing the results of the Y-maze behavioral test of mice in Example 2 of the present invention;

[0023] Figure 4 This is a graph showing the test results of a mouse novel object recognition experiment in Example 2 of the present invention.

[0024] Figure 1-Figure 4 In the horizontal axis, PBS: phosphate buffered saline, phosphate buffered saline; C: C is control, representing the control group without high temperature and high humidity environment stimulation; H: HTHH abbreviation, high temperature and highhumidity, representing the experimental group stimulated by high temperature and high humidity environment.

[0025] Compared with the C+PBS blank control group, P less than 0.05 indicated a statistically significant difference, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with the examples. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0027] Example 1

[0028] The anxiety and memory impairment mouse model was established as follows:

[0029] (1) Adaptive feeding: 20 eight-week-old adolescent mice were randomly divided into two groups, 10 mice in each group, and then placed in an SPF-grade experimental animal room with 5 mice in each cage for adaptive feeding. The feeding environment temperature was set at 24°C and the humidity was set at 50% RH. The light was on from 8:00 am to 8:00 pm and the darkness was on from 8:00 pm to 8:00 am. The mice were fed for one week. During the adaptive feeding process, both groups of mice had free access to the same feed and drinking water.

[0030] (2) After the adaptive feeding in step (1), the two groups of mice were divided into a control group C and an experimental group H. The control group C was placed in an SPF-grade experimental animal room with an ambient temperature set at 24°C and a humidity set at 50% RH; the experimental group H was placed in an artificial climate box with a temperature set at 36°C and a humidity set at 80% RH. Both groups were housed in cages of 5 mice each and had free access to the same feed and water. The feeding was continued for four weeks.

[0031] (3) Behavioral testing of mice: The mice in the control group C and the experimental group H fed in step (2) were subjected to elevated plus maze and open field tests, respectively.

[0032] Elevated plus maze behavioral test: The mouse was placed in the center of the plus maze with its head facing the open arm. The timer was started from the moment the mouse entered the maze. The instrument automatically monitored the number of times and time the mouse entered the open arm within 5 minutes. The monitoring data results are shown in Table 1 and plotted. Figure 1 The monitoring results show that the number of times the mice in the experimental group H entered the open arm was 7.86±1.335, and the number of times the mice in the control group C entered the open arm was 11.8±0.867. There was a significant difference between the number of times the experimental group H entered the open arm and the control group C (Pvalve=0.006); the time for the mice in the experimental group H to enter the open arm was 50.874±10.251s, and the time for the mice in the control group C to enter the open arm was 82.040±5.932s. There was a significant difference between the time of the open arm in the experimental group H and the control group C (Pvalue=0.009), indicating that the mice in the experimental group H were more anxious than the mice in the control group C.

[0033] Behavioral testing of the open field experiment: The mouse was placed in the center of the open field. The timer was started from the moment the mouse entered the open field. The instrument automatically monitored the time the mouse spent in the center of the open field and the distance it moved in the open field within 10 minutes. The monitoring data results are shown in Table 1 and plotted. Figure 2 The monitoring results showed that the mean time of the mice in the center of the open field in the experimental group H was 6.383±2.594s, and the mean time of the mice in the center of the open field in the control group C was 113.228±21.381s. There was a significant difference in the time spent in the center of the open field between the experimental group H and the control group C (Pvalue<0.0001); the mean movement distance of the mice in the open field in the experimental group H was 5815.800±631.412s, and the mean movement distance of the mice in the control group C was 8311.396±698.819s. There was a significant difference in the movement distance in the open field area between the experimental group H and the control group C (Pvalue=0.002).

[0034] This shows that the mouse anxiety model of the present invention was successfully established.

[0035] Example 2

[0036] Step (1) and step (2) are the same as those in Example 1 and will not be described in detail.

[0037] Step (3) The behavioral testing of mice was performed using Y-maze and novel object recognition.

[0038] Y-maze behavioral test: Put the mouse head down at the junction of the two arms of the Y-maze, start timing from the moment the mouse enters the Y-maze, monitor the order in which the mouse enters each arm within 8 minutes, if it does not enter the same arm three times in a row, it is recorded as correct, otherwise it is the opposite, and then record the number of spontaneous alternations. According to the recorded number of spontaneous alternations, calculate: spontaneous alternation rate (%) = [(spontaneous alternation number) / (total number of arm entries-2)] × 100%. The monitoring results data can be found in Table 1, and plotted Figure 3 The results showed that the number of spontaneous alternations of the experimental group H entering different arms three times in a row was 8.86±2.521, and the number of spontaneous alternations of the control group C entering different arms three times in a row was 15.90±1.588. There was a significant difference in the number of spontaneous alternations between the experimental group H and the control group C (P value = 0.003). It was found that the spontaneous alternation rate of the experimental group H entering different arms three times in a row was 52.057%±11.583%, and the spontaneous alternation rate of the control group C entering different arms three times in a row was 53.691%±3.371%. Compared with the experimental group H and the control group C, the experimental group H had a decreasing trend (P = 0.539).

[0039] Behavioral test of novel object recognition: On the first day, mice were placed in a test room (50×50×40 cm) without objects. 3 square space) for 10 minutes; on the second day, two identical original objects were placed in the test room, and the video was collected for 10 minutes, and then the mice were returned to the cage; on the third day, the mice were returned to the test room and stayed in the presence of an original object and a new object for 10 minutes. The instrument automatically monitored the number and time of the mouse's exploration of the new object and the original object within 10 minutes. The new object discrimination index was calculated based on the monitored exploration time of the new object and the exploration time of the old object. The new object discrimination index (%) = new object exploration time / (new object exploration time + original object exploration time) × 100%. When the discrimination index is greater than 50%, it indicates that the animal pays more attention to the new object. The monitoring result data is shown in Table 1, and it is plotted. Figure 4 .

[0040] The original object was a 2 cm2 cm×2 cm cubic wooden block, and the new object was a 2.5 cm2×2 cm triangular prism.

[0041] The results showed that the number of explorations of the experimental group H was 9.57±2.091, and the number of explorations of the control group C was 14.50±5.043. Compared with the control group C, the experimental group H had a decreasing trend (P=0.322); the discrimination index of the experimental group H was 0.626±0.261, and the discrimination index of the control group C was 0.564±0.877. There was no significant difference between the experimental group H and the control group C (P=0.614);

[0042] It can be seen that the memory function of the mice in the experimental group H was impaired, indicating that the mouse memory impairment model of the present invention was successfully established.

[0043] Table 1: Behavioral monitoring data statistics

[0044]

[0045] In summary, the method for constructing a mouse model provided by the present invention belongs to modeling in environmental stimulation, obtains experimentally verified results, and successfully establishes a mouse memory impairment model and a mouse anxiety model.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for simultaneously constructing an anxiety and memory impairment mouse model, characterized in that: The method comprises the following steps, (1) Adaptive feeding: The mice were randomly divided into two groups for adaptive feeding. The two groups were fed under the same feeding conditions, with a feeding environment temperature of 22-26°C, a humidity of 40-60% RH, and light and darkness alternating for 12 h each. The feeding was continued for one week. (2) The two groups of mice after adaptive feeding in step (1) are divided into a control group and an experimental group. The control group is raised in an environment with a temperature of 22-26° C. and a humidity of 40-60% RH, and the experimental group is raised in an environment with a temperature of 33-36° C. and a humidity of 80-95% RH, with light and darkness alternating for 12 hours each, and the continuous raising time is at least four weeks; (3) Behavioral testing of mice: The mice in the control group and the experimental group fed in step (2) were subjected to behavioral testing of elevated plus maze, open field test, Y maze and novel object recognition test, respectively.

2. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: The adaptive feeding in step (1) and the feeding environment of the control group in step (2) are both SPF-grade experimental animal rooms; the feeding environment of the experimental group in step (2) is an artificial climate box.

3. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: During the adaptive feeding in step (1) and the feeding of the control group and the experimental group in step (2), the mice were free to eat and drink, and the feed and drinking water used by the two groups of mice were kept consistent.

4. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: In step (3), the behavioral test of the elevated plus maze is specifically to place the mouse in the center of the plus maze with its head facing the open arms, and start timing from the time the mouse enters the maze. The monitoring instrument automatically monitors the number of times and time the mouse enters the open arms within 5 minutes.

5. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: In step (3), the behavioral detection of the open field experiment is specifically to place the mouse in the center of the open field, start timing from the time the mouse enters the open field, and the monitoring instrument automatically monitors the time the mouse spends in the center of the open field and the distance it moves in the open field within 5 minutes to 10 minutes.

6. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: In step (3), the Y-maze behavioral test is specifically to place the mouse head down at the junction of the two arms of the Y-maze, start timing from the moment the mouse enters the Y-maze, and monitor the order in which the mouse enters each arm within 5-10 minutes. If the mouse does not enter the same arm three times in a row, it is recorded as correct, otherwise it is recorded as the opposite. Then the number of natural alternations is recorded.

7. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: In step (3), the behavioral test of new object recognition is specifically as follows: on the first day, the mice are habituated to the test room without objects for 5 minutes to 10 minutes, and then returned to the original breeding environment; on the second day, the mice are returned to the test room, and video is collected for 5 minutes to 10 minutes in the presence of two original objects, and then the mice are returned to the original breeding environment; on the third day, the mice are returned to the test room, and stay in the presence of one original object and one new object for 5 minutes to 10 minutes, and the instrument automatically monitors the number and time of the mouse exploring the new object and the original object within 5 minutes to 10 minutes.

8. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 7, characterized in that: The test chamber should be at least 50×50×40cm 3 The size of the square space is very different from the original object and the new object in shape.

9. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: The mice were 6-8 weeks old and in puberty.

10. The method for simultaneously constructing an anxiety and memory impairment mouse model according to claim 1, characterized in that: The illumination time in step (1) is from 8:00 am to 8:00 pm, and the night time is from 8:00 pm to 8:00 am.

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