A method for constructing a social stress-based ad mouse depression comorbidity model and application

Abstract

The application discloses a kind of based on social stress AD mouse depression comorbidity model construction method and application, belong to biological medicine technical field.The CD-1 mouse with attack is placed in standard cage and is raised, 3xTg mouse is transferred to the side of standard cage with CD-1 mouse;Remove standard cage baffle, let CD-1 mouse hit 3xTg mouse, then put back baffle, CD-1 mouse is separated from 3xTg mouse with baffle, and 24 hours of social frustration threat is carried out, while combining despair stimulus and chronic mild unpredictability mild stimulation, repeated hitting and stimulation operation 8 days, and 3xTg mouse appears depression behavior related phenotype, and the depression comorbidity model of AD mouse is obtained.The method is simple to operate, does not need drug induction, the success rate of modeling is higher, the mortality is lower, and there are various social and psychological stress stimuli for mouse, can better simulate the occurrence of depression related to social factors, can obtain the mouse model suitable for studying the relevance of depression and AD.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a construction method and application of an AD mouse depression comorbidity model based on social stress. BACKGROUND

[0002] About 4.4% of the world's population has emotional disorders related to depression, and depression is often accompanied by mild cognitive impairment, which further aggravates the patient's condition and seriously affects the quality of life. Among them, geriatric depression disorder is a mental illness with a high incidence in the elderly population, which refers to the depression of the elderly with an onset age of 60 years old and above. Cognitive impairment is a common symptom of geriatric depression disorder. A prospective study found that 70% of elderly people with depression disorder were at risk of dementia, and many prospective studies found that elderly people with depression were 2-5 times more likely to develop cognitive impairment than the control group. The above studies suggest that depression disorder is an important risk factor for cognitive impairment in the elderly.

[0003] Although the comorbidity of depression and Alzheimer's disease (AD) is widely recognized in clinical practice, the relationship between the two is still unclear. Studies have found that depression may be both a risk factor for AD and an early symptom of AD. In order to better study the neural circuits and molecular mechanisms of depression and AD comorbidity, an ideal and stable animal model of depression and AD comorbidity is urgently needed. SUMMARY

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a construction method and application of an AD mouse depression comorbidity model based on social stress, in order to facilitate the study of the neural circuits and molecular mechanisms of depression and AD comorbidity.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0006] The first invention point of the present application provides a construction method of an AD mouse depression comorbidity model based on social stress, comprising the following steps:

[0007] 1) Place the aggressive CD-1 mice in a standard cage and feed them, and transfer the 3xTg mice to one side of the standard cage with CD-1 mice; the standard cage is obtained by inserting a transparent baffle in the middle of a common mouse feeding cage;

[0008] 2) Remove the transparent baffle with holes from the standard cage, let the CD-1 mice hit the 3xTg mice, and after the hitting is finished, put back the transparent baffle, separate the CD-1 mice from the 3xTg mice with the transparent baffle, and perform 24 hours of social defeat threat;

[0009] 3) repeat step 2) for 8 days, while combining different behavioral despair stimuli and chronic mild unpredictable stressors every day;

[0010] 4) perform depression behavior test on the 3xTg mice, and the mice with depression behavior related phenotypes are AD mouse depression comorbidity models.

[0011] Preferably, in step 1), the selection of the aggressive CD-1 mice is as follows: the CD-1 mice are bred under the condition of free access to food and water, and are adapted to the modeling environment for one week before screening; when screening, the test C57 mice are randomly selected to receive the beating of the CD-1 mice, and the CD-1 mice that can continuously beat the C57 mice are the aggressive CD-1 mice.

[0012] Further preferably, in the three 5-minute screening processes every day, the time interval between the two consecutive attacks is less than 60s, and the CD-1 mice must continuously attack the test C57 mice for at least 10s.

[0013] Preferably, in step 2), the CD-1 mice beat the 3xTg male mice four times a day, and each continuous beating of 10s is counted as one valid beating.

[0014] Preferably, in step 4), the depression behavior test includes a sugar water test, an open field test, a tail suspension test and a forced swimming test.

[0015] Further preferably, in the sugar water test, the 3xTg mice have decreased sugar water preference; in the open field test, the 3xTg mice have reduced time and distance in the central area; in the tail suspension test and the forced swimming test, the 3xTg mice have prolonged immobility time.

[0016] Preferably, the CD-1 mice are male mice with mating experience and the age is between 6 and 8 months.

[0017] Preferably, the 3xTg mice are 2-3-month-old male mice and carry three human AD-related gene mutations; the three AD-related genes are Psen1, APPSwe and tauP301L.

[0018] The second inventive point of the present application provides an application of the animal model obtained by the above-mentioned method for constructing an AD mouse depression comorbidity model based on social stress in the research of the correlation between depression and AD.

[0019] Preferably, the animal model is a model for researching the correlation between depression and AD through neural circuits and molecular mechanisms.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The application provides a construction method of an AD mouse depression comorbidity model based on social stress. 2, in the stimulation mode: adopt social failure combined with behavior despair stimulation and chronic mild unpredictable mild stimulation as double stimulation, a variety of social and psychological stimulation is carried out on the 3xTg mouse which has not yet appeared pathological and behavioral changes in early stage, which can effectively reduce traumatic stimulation, and can better simulate the occurrence of depression related to social factors. 3, in the construction method: simple operation, without any drug induction, short time consumption, depression phenotype can be induced by short-term behavioral stimulation. After stimulation intervention, the depression model in the early stage / latent period of AD can be efficiently and stably constructed through evaluation. The detection method is convenient, and can comprehensively and accurately evaluate whether the model is successful from the aspects of behavior, tissue morphology and physiological function. In summary, the AD and depression comorbidity animal model obtained by the method is a relatively suitable mouse model for studying the correlation between depression and AD, the success rate of modeling is high, and 75% of 3xTg mice with depression behavior can be stably obtained, the mortality is low, and the mouse is subjected to various social and psychological stress stimulation, which can better simulate the occurrence of depression related to social factors, is suitable for studying the cognitive state change and related pathological change of depression AD mouse, and is a relatively ideal and stable animal model for studying the neural circuit and molecular mechanism of depression and AD comorbidity.

[0022] Further, the selected CD-1 mouse is a male mouse with mating experience, which can frequently exhibit obvious aggressive behavior, and is more beneficial to the establishment of the model. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A is an acrylic transparent baffle in the social failure experiment; B is a toothbrush used in chronic mild unpredictable stimulation; C is a tail clamping tool (3# clip) used in chronic mild unpredictable stimulation;

[0024] Figure 2 It is a schematic diagram of the distribution position of the intruder mouse and the CD-1 attacking mouse in the modeling process;

[0025] Figure 3 It is a flow chart of the behavioral experiment for evaluating whether the animal model is successfully established;

[0026] Figure 4 Figure results of the mouse behavior and immunofluorescence staining verification experiment; wherein, A is the proportion of time that the mouse stays in the center area in the open field experiment, B is the proportion of time that the mouse stays still in the tail suspension experiment, C is the proportion of time that the mouse stays still in the forced swimming experiment, D is the sugar water preference index of the mouse in the sugar water preference experiment, E is the proportion of time that the mouse stays in the target quadrant in the Morris water maze test stage of the 3xTg mouse, F is the Iba-1 and Aβ immunofluorescence staining of the hippocampus of the mouse, G is the number of microglia cells in the hippocampus of the mouse, and H is the Tau protein phosphorylation staining of the neurons in the hippocampus of the mouse. DETAILED DESCRIPTION

[0027] The application will be further described in detail below with reference to the accompanying drawings:

[0028] The application provides a construction method of an AD mouse depression comorbidity model based on social stress, and the specific establishment process is as follows:

[0029] I. Establishment of an AD mouse depression comorbidity model based on social stress

[0030] 1) Selection of CD-1 attacking mice: a. CD-1 mice (male, 6-8 months old, with mating experience) are bred under the condition that food and water can be obtained at will, and need to be adapted to the modeling environment for one week before selection; b. randomly select one 8-20 week old C57BL / 6J male mouse as a "selector", and leave the CD-1 mice that can continuously attack the "selector"; c. use different "selector" mice for selection for three consecutive days (once a day), and the selection standard is that the time interval of continuous attack must be less than 60 s for two consecutive times in three 5 min selection processes, and the CD-1 mouse must attack the "selector" for at least 10 s, and the mouse that meets the above conditions is the CD-1 attacking mouse.

[0031] 2) Place the CD-1 attacking mouse selected in step 1) in a standard cage on one side for breeding, and the standard cage is obtained by inserting a detachable perforated acrylic transparent baffle in the middle of a common mouse breeding cage to separate the common mouse breeding cage into two spaces, and the acrylic transparent baffle is shown in Figure 1 A.

[0032] 3) see Figure 2The invader mice are 3xTg modeling group mice or C57BL / 6J modeling group mice, the 3xTg mice are 2-3 month-old male 3xTg transgenic mice purchased from Jackson Laboratory, USA, and the mice simultaneously carry three AD-related genes of human: APP K670_M671delinsNL (Swedish), MAPT P301L, PSEN1 M146V.

[0033] 4) Remove the acrylic transparent baffle, and let the CD-1 attack mice hit the corresponding invader mice, and each invader mouse is hit a total of four times, and each continuous hit for 10s is counted as one valid hit.

[0034] 5) After the hitting is completed, the acrylic transparent baffle is put back, the CD-1 attack mice and the corresponding invader mice are separated by the acrylic transparent baffle, and a social frustration threat for 24 hours is performed.

[0035] 6) After the 24-hour social frustration threat, the CD-1 attack mice are replaced, and the above steps 4) and 5) are repeated for 8 days, while different behavioral despair stimuli and chronic mild unpredictable mild stimuli are combined every day, and the specific stimuli are shown in Table 1:

[0036] Table 1 Different behavioral despair stimuli and chronic mild unpredictable mild stimuli

[0037]

[0038]

[0039] 7) After the last modeling, all invader mice are individually placed, and the depression behavior of the 3xTg mice is detected, and the evaluation criteria for depression behavior are that the sugar water preference index of the mouse sugar water preference experiment is ≤80%, the proportion of time spent by the mouse in the central area in the open field experiment is ≤10%, the proportion of time spent by the mouse in the tail suspension experiment is ≥60%, and the proportion of time spent by the mouse in the forced swimming experiment is ≥65%, and the model mice at least meet three of the above criteria, that is, the AD mouse depression comorbidity model based on social stress is successfully constructed, and the mice with depression behavior related phenotypes are the AD mouse depression comorbidity model based on social stress.

[0040] II. Verification of comorbidity animal model

[0041] The behavioral index analysis related to the application is analyzed by using a SMART 3.0 system developed by Panlab, which is widely used in medical experiments and neural research fields, and the SMART can provide functions of trajectory tracking, three-point analysis, activity analysis, event recording and social activity.

[0042] Referring to Figure 3 In the behavioral experiment sequence diagram, 24 hours after the last modeling, the sugar water, open field, forced swimming, tail suspension and cognitive behavior experiment of Morris water maze are performed on the two groups of mice (3xTg modeling group and 3xTg control group), and the immunofluorescence staining experiment is performed four months later. If the sugar water experiment mouse sugar water preference decreases significantly, the open field experiment mouse time and distance in the central area significantly decrease, the tail suspension and forced swimming experiment shows that the mouse resting time significantly extends, the Morris water maze experiment shows that the mouse time ratio in the target quadrant significantly decreases, and the immunofluorescence staining experiment four months after modeling shows that the 3xTg mouse will deposit Aβ amyloid protein, indicating that the modeling is successful. The specific steps are as follows:

[0043] Experimental materials: sugar water experiment bottle, 40cm*40cm*15cm open field box; standard tail suspension box; forced swimming bucket diameter 10cm, height 25cm, transparent.

[0044] Experimental animals: 3xTg control group mice, 3xTg modeling group mice.

[0045] Experimental method:

[0046] The sugar water preference experiment steps are as follows: each mouse is individually fed during the entire experiment, and two bottles of 1.5% sucrose solution are provided, and the mice are not fasted for 24h; then the sucrose water is replaced with two bottles of pure water, and the mice are not fasted for 24h; the mice are allowed to rest for 12h; after resting, the mice are fasted for 24h, and then each mouse is provided with one bottle of 1.5% sucrose water and one bottle of pure water, and the mice are allowed to drink freely. After 24h, the water bottles are removed, and the sucrose water consumption is calculated as the ratio of the total water consumption, that is, the sucrose preference index of the mouse.

[0047] The open field experiment steps are as follows: the open field box is sprayed with 75% alcohol and cleaned to ensure that there is no odor and foreign matter. The mice in each group are placed in the open field box with their backs to the wall, the curtains are pulled up to prevent external environment from interfering with the mice, the video recording system is turned on, and the exploration trajectory of the mice in the open field box is recorded for 10min. After the video recording is completed, the mice are taken out and placed back in the original feeding environment, and then the open field box is sprayed with 75% alcohol and cleaned before the next experiment.

[0048] The forced swimming test procedure is as follows: before starting, prepare a suitable size transparent round water bucket, add water to the water depth of about 20 cm. Put the mice in each group into the water respectively, turn on the video equipment, record the activity of the mice in the water for 5 min, then take out the mice and dry them, and put them back into the feeding cage to rest. The time of the mice floating on the water surface without moving within 5 min is counted.

[0049] The tail suspension test procedure is as follows: at the beginning, the tails of mice in each group are taped with adhesive tape, and the mice are hung on the tail suspension device. A plastic tube is placed around the mouse's tail to prevent the mouse from climbing upwards during the suspension process. The mouse's head should be about 10 cm away from the tray at the bottom of the device. Turn on the video recording system and record for 8 min. The immobile time of the mouse in the last 6 min is counted.

[0050] The Morris water maze test procedure is as follows: before starting the water maze test, prepare a water pool with a diameter of 120 cm and a platform with a diameter of 10 cm. Fill the water pool with water until the platform is submerged. Add whitening agent to the water. The test is conducted for 5 days. Days 1-4 are the positioning navigation test. The water pool is divided into four quadrants, and the platform is placed in the fourth quadrant. Each group of mice is allowed to swim for 1 min. If the mouse finds the platform within 1 min, the test is ended. If the mouse does not find the platform, the mouse is guided to the platform location for 5-7 s. Each experimental mouse is placed into the water pool from one of the four quadrants once. The video recording is repeated for 4 days. The last day is the spatial exploration test. On the test day, remove the platform. Each mouse is placed into the water pool from one of the four quadrants once. The mouse is allowed to swim for 1 min. The video recording is used to count the data.

[0051] The immunofluorescence staining test procedure is as follows: perfuse and fix the brains of the mice in each group with paraformaldehyde. Take out the brain tissue and immerse it in 4% PFA at 4°C overnight for post-fixation. Then replace it with 30% sucrose solution at 4°C for 48 hours or more for dehydration. When the tissue is completely submerged, prepare frozen sections with a thickness of 20 μm. To prevent the sections from falling off the glass slide, fix the sections with 4% PFA at room temperature for 30 minutes and wash them with PBS for 3 x 10 minutes. Incubate the sections with blocking solution (3% BSA + 0.3% Triton) in a wet box at room temperature for 1 hour. Add the primary antibody (microtubule-associated protein Tau aggregates, AT8, monoclonal antibody synthesized by Wuhan Jin Kai Rui Biological Engineering Co., Ltd.; beta-amyloid, Aβ17-24, monoclonal antibody purchased from BioLegend, USA; rabbit anti-microglia / macrophage-specific protein, Iba-1, polyclonal antibody purchased from Wako, Japan; neuron marker, NeuN, polyclonal antibody purchased from Millpore, USA) prepared with blocking solution in a wet box at room temperature overnight. The next day, wash the sections with PBS for 3 x 10 minutes, add secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG, purchased from Invitrogen, USA) in a wet box at room temperature for 1 hour, and then wash them with PBS for 3 x 10 minutes. Add DAPI (4', 6'-diamidino-2-phenylindole, purchased from Invitrogen, USA) in a wet box at room temperature for 10 minutes, and then wash them with PBS for 3 x 10 minutes. Mount the sections on glass slides with antifade mounting medium (purchased from Invitrogen, USA) and seal them with nail polish. Use a fluorescence microscope to observe and photograph the sections. 594-labeled donkey anti-mouse polyclonal secondary antibody, Alexa Donkey anti-rabbit polyclonal secondary antibody labeled with 647 was purchased from Cell Signaling, USA. The cells were incubated at room temperature for 2 h and washed with PBS 3 × 10 min. The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole, DAPI) purchased from Sigma, Germany for 5 min. The cells were then mounted with 50% glycerol. The cells were observed and images were acquired using a laser confocal microscope.

[0052] See the experimental results. Figure 4 ,Depend on Figure 4 As can be seen from A, the proportion of time spent in the central region and the distance traveled by mice in the 3xTg model group were significantly reduced; Figure 4 As can be seen from B, the proportion of rest time in the tail suspension test was significantly increased in the 3xTg model group mice; Figure 4 As can be seen from C, the proportion of still time in the forced swimming test was significantly increased in the 3xTg model group mice; Figure 4 As can be seen from D, the preference for sucrose water in the 3xTg model group mice was significantly reduced; Figure 4 As can be seen from E, the proportion of time spent by 3xTg mice in the target quadrant decreased during the Morris water maze test; from Figure 4 As can be seen from the middle F, Aβ deposition is observed in neurons of the hippocampus of 3xTg mice; by Figure 4 As can be seen from G, the number of microglia in the hippocampal neurons of 3xTg mice is significantly increased; Figure 4 As shown in Figure H, elevated levels of Tau protein phosphorylation were observed in neurons of the mouse hippocampus (scale bar = 50 μm). Based on these results, it can be concluded that a mouse model of AD comorbid depression based on social stress has been successfully established.

[0053] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for constructing a social stress-based AD mouse model of depression comorbidity, characterized in that, The method comprises the following steps: 1) placing the aggressive CD-1 mice in a standard cage, and transferring the 2-3-month-old 3xTg mice to one side of the standard cage with the CD-1 mice; the standard cage is obtained by inserting a transparent baffle in the middle of a common mouse breeding cage; 2) removing the transparent baffle with holes of the standard cage, and allowing the CD-1 mice to hit the 3xTg mice, wherein the CD-1 mice hit the 3xTg mice four times a day, and each continuous hitting for 10 s is counted as one valid hit; after the hitting is completed, the transparent baffle is put back, and the CD-1 mice and the 3xTg mice are separated by the transparent baffle, and the social frustration threat is performed for 24 hours; 3) repeating the operation in step 2) for 8 days, while combining different behavioral despair stimuli and chronic mild unpredictable mild stimuli every day; 4) detecting the depression behavior of the 3xTg mice, and the mice with the depression behavior related phenotype are obtained as the AD mouse depression comorbidity model.

2. The method according to claim 1, wherein, In step 1), the selection method of the aggressive CD-1 mice is as follows: the CD-1 mice are bred under the condition that food and water can be obtained at will, and are adapted in the modeling environment for one week before screening; when screening, the test C57 mice are randomly selected to receive the hitting of the CD-1 mice, and the CD-1 mice that can continuously hit the C57 mice are the aggressive CD-1 mice.

3. The method according to claim 2, wherein the social stress-based AD mouse model of depression comorbidity is constructed by, In the three 5-min screening processes every day, the time interval of continuous hitting is less than 60 s, and the CD-1 mice must continuously hit the test C57 mice for at least 10 s.

4. The method according to claim 1, wherein, In step 4), the depression behavior detection includes a sugar water experiment, an open field experiment, a tail suspension experiment and a forced swimming experiment.

5. The method according to claim 4, wherein the social stress-based AD mouse model of depression comorbidity is constructed by, In the sugar water experiment, the sugar water preference of the 3xTg mice decreases; in the open field experiment, the time and distance of the 3xTg mice in the central area decrease; in the tail suspension experiment and the forced swimming experiment, the resting time of the 3xTg mice is prolonged.

6. The method according to claim 1, wherein, The CD-1 mice are male mice with mating experience, and the age is between 6 and 8 months.

7. The method according to claim 1, wherein the social stress-based AD mouse model of depression comorbidity is constructed by, The 3xTg mice carry mutations of three AD-related genes of human origin, and the three AD-related genes are Psen1, APPSwe and tauP301L.

8. The animal model obtained by the method for constructing the AD mouse depression comorbidity model based on social stress according to any one of claims 1-7 is used in the research on the correlation between depression and AD.

9. Use according to claim 8, characterized in that, The animal model is a model for researching the correlation between depression and AD through neural circuits and molecular mechanisms.

Images