CN109512819B - Autism mouse model, construction method and application thereof - Google Patents
Autism mouse model, construction method and application thereof Download PDFInfo
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Abstract
The application discloses an autism mouse model, a construction method and application thereof. The construction method comprises the step of carrying out pregnancy intervention on a pregnant mouse by using a rapamycin medicament so as to obtain an autism filial generation mouse model. The invention establishes a new reliable animal model for ASD related research and finds a new direction for preventing and treating autism in the future.
Description
Technical Field
The disclosure relates to a mouse model, a construction method and application thereof, in particular to an autism mouse model, a construction method and application thereof.
Background
Autism Spectrum Disorder (ASD) is a common Neurodevelopmental Disorder caused by a developmental Disorder of the nervous system. According to Baird, G[1]A survey in 2006 showed an incidence of about 1% in the global adolescence of ASD; according to American Psychological Association (APA) modifications made in 2013, ASD patients often manifest impaired social, language handicap (language lag to speech loss), repetitive and/or compulsive behavior and speech mimics, hyperactivity disorders, memory deficits, learning, motor skills or other abnormal excitation of neurological functions, and ultra-or hypo-excitabilitySensitive sensory irritation, anxiety, adaptation to new environments and habituation difficulties. Therefore, ASD patients place a great burden on the home as well as the whole society.
Despite such high prevalence and great harm to ASD, its etiology remains unclear. There are reports demonstrating that rapamycin and its analogs have therapeutic effects on autism.
One key problem encountered during ASD studies is the lack of simple easily reproducible animal models. Many research institutes are dedicated to basic experiments on autism and autism-like behavior, and therefore there is a strong need for a stable experimental animal model with an exact mechanism. Now ASD studies rely mainly on mutation or knock-out models of a single gene based on genetic factors. However, such models often differ in phenotype according to mutation or knock-out of different genes. In addition, the period required for the propagation of the model is long, and the propagation base number of the required mice is large. This presents a barrier to ASD research, particularly ASD drug development. Therefore, development of an animal model of ASD based on a certain mechanism is imminent.
mTOR (catalytic target of rapamycin) is involved in regulating multiple cellular processes from protein, lipid synthesis to autophagy (autophagy)[2,3]. mTOR is also involved in the development of various nervous system diseases[3]. Rapamycin (rapamycin) is a selective inhibitor of the mTOR receptor[4-8]。
Disclosure of Invention
In view of the above-mentioned deficiencies or inadequacies in the prior art, it would be desirable to provide an ASD model that is simple and easy to replicate.
The inventor discovers for the first time that: when rapamycin is contacted during pregnancy, the offspring can generate autism-like behaviors, thereby providing a new method for constructing an autism model.
The invention uses rapamycin medicine to intervene pregnant period of pregnant mouse, thus obtaining autism filial generation mouse model.
According to one aspect of the invention, a construction method of an autism mouse model is provided, and the construction method comprises the step of carrying out pregnancy intervention on pregnant mice by rapamycin, so as to obtain an autism progeny mouse model.
According to one embodiment, a rapamycins solution is injected intraperitoneally at 3-7 mg/kg body weight starting at 11, 12 or 13 days of pregnancy for female healthy mice in the sexual maturity stage until delivery of the pregnant mice. Preferably, the injection is initiated at 12 days of gestation. Preferably, the rapamycin solution is injected intraperitoneally at 5mg/kg body weight until delivery of pregnant mice.
According to one embodiment, the mouse is preferably C57 BL/6.
According to one embodiment, the autism mouse model is constructed by injecting lapachycin solution intraperitoneally at 5mg/kg body weight starting at 12 days of pregnancy for C57BL/6 mice in the sexual maturity stage until delivery of the pregnant mouse.
According to one embodiment, the rapamycin is dissolved in dimethyl sulfoxide (DMSO).
According to another aspect of the present invention, there is provided an autism mouse model constructed according to the method described above. According to one embodiment, the model is used to study the pathogenesis of autism.
According to another aspect of the invention, the use of the mouse model for treating autism for screening a therapeutic agent for treating autism is provided.
According to another aspect of the invention, the use of the mouse model for autism for studying the pathogenesis of autism is provided.
The general state of the pregnant mouse in the whole pregnancy and the related functions of the offspring mouse after birth are monitored, so that the fact that the contact of rapamycin medicaments in the pregnancy can cause a series of movement, social interaction and cognitive dysfunction of the offspring shortly after birth, namely, the autism-like expression appears is proved, a novel reliable animal model is established for the related research of ASD, and a novel direction is explored for preventing and treating autism in the future.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:
FIG. 1 shows body sizes or brain volume sizes of offspring mice of experimental and control groups;
figures 2 to 12 show the results of the behavioural test in terms of mouse autism-like social behaviour; wherein fig. 2 to 5 show the results of a three-compartment social experiment in which "mouse" indicates a side of a mouse cage in which an isosexual mouse is placed, "empty mouse cage" indicates a side in which an empty mouse cage is placed, "familiar mouse" indicates a side in which a familiar mouse is placed, "unfamiliar mouse" indicates a side in which an unfamiliar mouse is placed; FIGS. 6 and 7 show the results of open field experiments in which the "central region" represents activity in the central region of the open field and the "peripheral region" represents activity in the peripheral region of the open field; FIG. 8 shows the results of a social interaction experiment; fig. 9 shows the results of a juvenile play experiment; FIG. 10 shows the results of nesting experiments; FIG. 11 shows the experimental results of the spontaneous selection of the Y maze; fig. 12 shows the results of the self-stroking experiments.
Figures 13 to 20 show the results of the behavioural tests on cognitive behaviour in mice; wherein P <0.05, P < 0.01, P < 0.001; FIGS. 13 and 14 are contents of experiments of new object recognition, wherein FIG. 13 shows a comparison of exploration new object bias in new object recognition; FIG. 14 shows a comparison of new object recognition memory; FIGS. 15 to 20 show the contents of the water maze experiment, in which FIG. 15 shows the comparison of the positioning flight evasion latencies of the water maze mice; FIG. 16 is a comparison of search distances for water maze mouse positioning navigation training; FIG. 17 is a comparison of the number of times of traversing the platform in the water maze space exploration experiment; FIG. 18 is a comparison of water maze positioning navigation training swimming speeds; FIG. 19 is a comparison of residence times in target quadrants of a water maze space exploration experiment; FIG. 20 is a typical illustration of the swimming trajectory of a water maze space exploration experimental mouse.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
The inventor discovers for the first time that: if rapamycin is contacted during pregnancy, the offspring can be caused to have autism-like behavior. Specifically, the invention uses rapamycin inhibitors to directly inhibit mTOR receptors, thereby generating a series of molecular mechanisms for activating autophagy pathways and proposing that excessive autophagy is one of the possible causes of autism and autism-like behavior.
Therefore, the invention provides a novel method for constructing the autism model.
According to one aspect, the invention provides a method for constructing an autism mouse model, which comprises performing pregnancy intervention on a pregnant mouse with a rapamycin medicament, thereby obtaining an autism progeny mouse model.
According to one embodiment, the mouse is preferably C57 BL/6. The gene information of the C57BL/6 mouse is clear, and the fertility is strong, so the method is widely applied to the research of serious diseases. According to one embodiment, other strains of mice may also be used, such as Balb/c, c3H, and the like.
According to one embodiment, a rapamycins solution is injected intraperitoneally at 3-7 mg/kg body weight starting at 11, 12 or 13 days of pregnancy for female healthy mice in the sexual maturity stage until delivery of the pregnant mice. Preferably, the injection is initiated at 12 days of gestation. The 12 days of pregnancy are selected because the initial time of the mouse brain cortical neuron development is about 11 days of pregnancy, while the 12 days of pregnancy is the optimal time of the cortical neuron development, and the injection is started from 12 days of pregnancy, so the stillbirth rate is lower. Preferably, the rapamycin solution is injected intraperitoneally at 5mg/kg body weight until delivery of pregnant mice. When the rapamycin solution is injected into the abdominal cavity at a concentration of more than 7mg/kg body weight, the death rate is high.
According to one embodiment, the rapamycin is dissolved using DMSO as a solvent. This is because rapamycin has low solubility and other solvents cannot dissolve. DMSO is a common universal solvent, and can well dissolve DMSO. Rapamycin was also dissolved in 0.02% alcohol, but it was not used because of its strong irritativeness.
According to one embodiment, 9-week-old female healthy C57BL/6 mice are intraperitoneally injected with a rapamycin solution (5mg/kg body weight) starting at 12 days of gestation until 18 days of gestation.
In order to verify whether the obtained offspring mice can be used as an autism model, the body types and the brain volume of the offspring mice are compared, and the social, motor and cognitive function behaviors of the offspring mice are tested.
Experimental materials and groups
The C57BL/6 mice were purchased from Beijing Huafukang Bio Inc., and were bred and bred in the laminar flow animal house of the neurosurgical institute of the eight brain hospitals affiliated with the general Hospital of the army. The indoor temperature is kept at 22 +/-2 ℃ throughout the year, the humidity is 55-65%, and the circadian rhythm of 12 hours indoors is kept every day (day 08:30-20: 30). All mice were given full-nutrition pellet feed and sterile water drinking and were periodically subjected to clean bedding changes. The related management of all experimental animals follows the animal experiment management regulation of Ministry of health of the people's republic of China, and the use and management of all experimental animals in the research are approved by the related experimental animal management committee.
Rapamycin was purchased from Sigma (Sigma Aldrich), No. V900930, specification 1 MG. The solvent was DMSO (dimethyl sulfoxide, Sigma Aldrich, D2650). 1mg of rapamycin was dissolved in 1mL of DMSO.
Female healthy C57BL/6 mice 9 weeks old were injected intraperitoneally with rapamycin solution (5mg/kg body weight) starting at 12 days of gestation, and the amount was measured based on the body weight of the mice. About 0.1-0.2 ml per mouse was injected until 18 days of pregnancy. The control group was injected with DMSO solvent.
FIG. 1 shows the body sizes or brain volume sizes of the offspring mice of the experimental and control groups, wherein the left side is the rapamycin group of the offspring mice and the right side is the control group of the offspring mice. As shown in FIG. 1, the offspring mice of the experimental group and the control group were P after birth0In days, mice injected with rapamycin were smaller than control mice, whether from body size or brain volume size.
The offspring mice of the experimental group and the control group are subjected to the behavioral tests of social, motor and cognitive functions
The main equipment used is as follows:
3-Chamber test laboratory Bioseb company (USA)
Y-maze test apparatus Bioseb company (USA)
Hot plate test apparatus Bioseb company (USA)
Light-dark box Experimental apparatus Bioseb company (USA)
Standard mouse rearing cage Zhuhai City Xin instrument Co., Ltd (China)
Blue Instrument Co Ltd of Water maze swimming pool, China department
Smart image acquisition System Panlab corporation (USA)
Beijing Huafukang Biotech GmbH Co Ltd for cleaning padding
Clean cotton pad Beijing Huafukang Biotech GmbH
Independent mouse cage feeding box Chinese V.M.I.S36
Darluan cheese biscuit food shares company of GmbH (China)
Beijing chemical industry Limited liability company of absolute ethyl alcohol
Experimental animals and behavioural procedures
1.1 Experimental animals
Offspring mice from 6-8 week old (Rapa: n ═ 7) and control (Con: n ═ 13) groups were used in a series of autism-related behavioral tests. In the feeding process of the mice used in the experiment, weaning breeding is given to weaning parents at 4 weeks after birth, and the mice are fed in cages according to sex at about 5 weeks after birth, and 3-4 mice with the same sex are bred in each cage. The feeding environment was as described above. Among them, the mice used in the juvenile play experiment (Juvenil play test) were all the mice of 21 days after birth.
1.2 preparation before experiment and behavioural flow
Before the ethological test is carried out, all the mice to be tested are numbered respectively, the feeding cages of all the mice to be tested are cleaned and padded for replacement one day before the ethological test is carried out, and all the mice to be tested are placed in a room for the ethological test so as to adapt to a new environment.
The behavioural process flow is as follows:
1. three-compartment social preference experiment (3-chamber social preference test)
2. Open field experiment (Open field test)
3. Social interaction test (regenerative interaction test)
4. Self-stroking experiment (Self-stroking test)
5. Y maze spontaneous selection experiment (Y-maze spontaneous alteration test)
6. Nesting experiments (Nest building test) were performed at night during the behavioural tests described above
7. The juvenile play experiment is carried out about 21 days after the test mice are born
8. Water Maze experiment (Morris Water Maze test)
All behavioral tests were performed during the day (09:00-17:00) (except nesting experiments).
1.3 results of the experiment
1. Mice injected with rapamycin showed autistic-like social dysfunction.
The effect of rapamycin injection on social behaviour in mice was first studied. In the experiment, the ethological test of autism-like social behaviors was performed on rapamycin-injected mice of 6-8 weeks of age (except for juvenile play experiments) and control DMSO-injected mice.
(1) Three-compartment social preference experiment: the three-chamber social preference experiment can reflect social interaction to a certain degree, a static empty cage is placed in a chamber (chamber) on one side to serve as object stimulation (non-social stimulation), and a cage filled with strange age-related mice is placed in a chamber on the other side to serve as social stimulation. The natural sociability of a mouse makes it more preferable to stay in and communicate more with the bedroom on the side of an unfamiliar mouse; if the mice have no obvious preference between strange and familiar mice, it can be indirectly stated that the mice have a corresponding barrier to social interaction.
(2) The open field experiment is also called open box experiment, and is a method for evaluating the autonomous behavior of an experimental animal in a new and different environment and researching the behavior and the tensity. The experiment reflects the autonomous behavior and the exploration behavior of the experimental animal in the strange environment according to the occurrence frequency, the duration and the like of certain behaviors of the experimental animal in the novel environment.
(3) Social interaction experiment: the social interaction experiment is another experiment reflecting social behaviors and whether the social behaviors are defective or not. Wherein the aggressive behavior comprises: the tested mouse actively attacks the bite/scratch interaction mouse, and the tested mouse and the interaction mouse mutually form the behaviors of standing-like angry and the like; non-aggressive behavior includes: the method comprises the following steps that a tested mouse actively chases an interactive mouse, the tested mouse actively trails the interactive mouse, the tested mouse actively approaches the interactive mouse and closely contacts with the body, the tested mouse actively takes forelimbs on the interactive mouse, the tested mouse actively brushes the hair of the interactive mouse, and the tested mouse actively contacts with the interactive mouse through the nose and the nose or contacts with the lower body.
(4) And (3) playing experiments of teenagers: juvenile play experiments are another behavioural test that tests early social dysfunction. The test content includes the time at which social activity, self-stroking, and mining occurred. Social behaviors include: the method comprises the following steps that a tested mouse actively chases an interactive mouse, the tested mouse actively trails the interactive mouse, the tested mouse actively approaches the interactive mouse and closely contacts with the body, the tested mouse actively takes forelimbs on the interactive mouse, the tested mouse actively brushes the hair of the interactive mouse, and the tested mouse actively contacts with the interactive mouse through the nose and the nose or contacts with the lower body; the self-stroking behavior comprises: the repeated actions of the tested mice spontaneously combing and/or licking any part of the body by the tongue; digging action, namely the action of digging the padding with forelimbs of the tested mouse.
(5) Nesting experiment
1. Taking a clean standard mouse rearing cage, and putting a cleaning pad with the thickness of about 2-3 cm into the standard mouse rearing cage for later use;
2. putting a cleaning cotton pad with the size of 5cm multiplied by 1cm into a squirrel cage to be used;
3. the test mice were placed in the prepared cages at 18:00 a day (one test mouse per cage) and given sufficient food and water, and then the experiment was performed at normal light rhythm (20: 30-8: 30 days with the light source off);
4. and (4) scoring the nests built by the tested mice on the next day of 8:40, wherein the nests are divided into 1-5 according to the shape and the height of the nested bodies. The specific criteria are as follows: the cotton pad was either barely used (more than 90% intact) or merely moved in position, giving 1 point; the cotton pad part is shredded (50-90% remains intact) for 2 minutes; most of the cotton pads are shredded, but the shredded cotton pads are only scattered and distributed for 3 minutes; most cotton pads are shredded, and the shredded cotton pads are intensively placed at a certain position (generally a certain corner) of a mouse cage, the placement shape of the shredded cotton pads can be roughly seen as a nest, but the shredded cotton pads are slightly flat, and a nesting mouse cannot be placed in the shredded cotton pads (the nesting height is 50% of the body height of the first mouse), and 4 minutes is given; more than 90% of the cotton pads are shredded, the shredded cotton pads are concentrated at a certain corner of the mouse cage and are placed into a nest shape, the nesting height is higher than 50% of the body height of the mouse, and the mouse can be well placed in the nest shape for 5 minutes;
5. and after the grading is finished, taking out the mouse to be tested, replacing the padding and the cotton pad completely, and testing the next mouse at night.
(6) Y maze spontaneous selection experiment
1. The test mouse was gently placed from the starting arm of the Y maze, allowed to freely select into the target arms on the left and right sides, and once selected, the door of the target arm was gently closed, and the test mouse was allowed to search for 1 minute in the target arm. The other arm which is not selected by the tested mouse at the moment is a non-target arm;
2. after the exploration is finished, the door of the target arm is opened, the tested mouse is taken out slightly, then the tested mouse is placed in the initial arm in sequence for 10 times, and the times of the tested mouse entering the target arm selected before in 10 times are recorded. Mice entered the standard: the double hind limbs and the caudal root cross the portal line. After each selection of the mouse to be tested, the mouse is allowed to enter the whole selected arm and stays in the selected arm for 5-10 seconds, and then is gently taken out for placing next time. Finally, the percentage of non-selection changes in each mouse tested was counted ((number of selection target arms/10) × 100);
3. after the test is finished, the mouse to be tested is taken out, then the Y labyrinth device is cleaned by 70% ethanol solution, and the test of the next mouse is carried out after the Y labyrinth device is dried.
Figures 2 to 12 show the results of the behavioural test in terms of mouse autism-like social behaviour; the results of the three-compartment social preference experiments are shown in fig. 2 to 5, where "mouse" indicates the side of the mouse cage with the same sex, "empty mouse cage" indicates the side with the empty mouse cage, "familiar mouse" indicates the side with the familiar mouse, and "unfamiliar mouse" indicates the side with the unfamiliar mouse. When stimulated with empty and strange mice as preferences, the rapamycin injected mice had no apparent preference for empty and strange mouse cages (t 0.471, p 0.646), although the communication time with strange mouse cages was greater than that with empty cages, the difference was not statistically significant (t 0.514, p 0.616), and the communication time difference was significantly reduced compared to DMSO injected control mice (t 3.791, p 0.001); not only did the rapamycin injected mice show no significant preference for the familiar and strange mice (t 0.284, p 0.781) and no significant difference in the communication time between the familiar and strange mice (t 0.101, p 0.922), but the communication time was still significantly reduced compared to the control DMSO injected mice (t 3.024, p 0.007). It can be seen that the mice in the rapamycin group exhibited significant social dysfunction compared to the control group.
The results of the open field experiments are shown in fig. 6 and 7, where "central region" indicates activity in the central region of the open field and "peripheral region" indicates activity in the peripheral region of the open field. As shown in the figure, the mice in the rapamycin-injected group had significantly less time to move in the central area of the open field than the control group, and had significantly more time to move in the area around the open field than the control group, and the difference was statistically significant (P < 0.05), demonstrating that the mice in the rapamycin-injected group lack normal exploratory behavior for the central area; in terms of movement distance, the rapamycin-injected mice are also significantly smaller than the control mice, the difference is statistically significant (P < 0.05), and more, the stroke is performed in the corners (self-stroking, etc.). The results of the open field test prove that compared with the control group, the rapamycin group mice lack interest in fresh things, are willing to move in own areas only and are unwilling to explore external unknown things, and typical carved repetitive actions such as stroking and the like also occur for a long time in the moving process of the areas around the open field.
The results of the social interaction experiment are shown in fig. 8. As shown in the figure: rapamycin injected mice were significantly lower than littermate control DMSO injected mice, although both in terms of time to onset of aggressive and non-aggressive behavior, and the differences were statistically significant (t 4.930, 4.145, p < 0.001). The results of this experiment again demonstrate that the social function of the mice in the rapamycin group is significantly deficient and that the mice in the rapamycin group are more prone to move independently than normal communication with other mice of the same age.
The results of the juvenile play experiment are shown in figure 9. Normally, the social behavior of the mice should be relatively long, and the self-stroking and digging behavior should be short, over a 10 minute test period. If mice with social dysfunction appear, the social time is obviously reduced, and the self-stroking behavior and the digging behavior are obviously increased. As shown in fig. 9, social activity occurred in 21 days postnatal mice injected with rapamycin significantly less than in their littermate control mice injected with DMSO (t-5.989, p < 0.001), although there was no significant difference in the time of occurrence of another repetitive behavior with psychoactive escape (digging behavior) (t-0.01, p-0.992), but the time of occurrence of repetitive self-combing behavior was significantly longer than in the DMSO-injected group of mice (t-7.387, p < 0.001).
Figure 10 shows the results of nesting experiments, which also show: rapamycin injected mice had a poorer nesting ability score than littermate control DMSO injected mice (t-3.380, p-0.003).
In addition, behavioral tests related to autism repeat stereotypical behavior were performed in mice injected with DMSO and rapamycin-injected mice injected with rapamycin and their littermate controls, showing that mice injected with rapamycin exhibited autism-like repeat stereotypical behavior. The specific results are shown in fig. 11 and 12:
figure 11 shows the experimental results of the spontaneous selection of the Y maze, showing: rapamycin injected mice have difficulty in altering the first selected target arm and thus exhibit a higher proportion of unchanged selections (No alteration (%) 57.14 ± 2.85; control group 49.23 ± 2.39);
fig. 12 shows the results of the self-stroking experiments, showing: rapamycin injected mice exhibited more repetitive stroking behavior than control DMSO injected mice (t-5.177, p < 0.001).
2. Rapamycin injected mice display cognitive dysfunction
The effect of rapamycin injection on cognitive function in mice was studied. The method comprises the following steps: new object identification test and water maze experiment.
(1) And performing a new object recognition ethology experiment on the offspring mice to test the non-spatial learning and memory capacity of the offspring mice, and further verifying the influence of the rapamycin on recognition memory. The test for recognition of new objects is an experiment designed by Ennaceur & Delaour (1988) based on the innate tendency of rodents to explore new environments, and the memory of the tested animals is evaluated according to the exploration time of the animals for familiar objects seen in the environment and for new and different objects never seen in the environment, and the recognition of memory is examined. Under normal conditions, the search time of a mouse for an unfamiliar object is often longer than that of a familiar object; if mice spend significantly less time (RI) exploring an unfamiliar object than normal mice, this contextual memory is suggested to be a barrier, i.e., impaired ability to distinguish between familiar and new foreign objects.
Figures 13 to 20 show that rapamycin injected mice show cognitive dysfunction. In which fig. 13 and 14 show the results of the new object identification test. As shown in fig. 13: during training, the preferences for two groups exploring the same object were (mean ± SEM): the rapamycin group was 0.48 + -0.008, the control group was 0.48 + -0.13, and the differences between the two groups were not statistically significant (p ═ 0.74), so there was no significant bias difference between the control group and the rapamycin injected group. These observations suggest that mice in both groups have the same level of motivation, curiosity and interest, exploring new subjects. After 30 minutes of training, a new object is replaced with an identified object. As shown in fig. 14: the positive control mice showed significant preference and new objects were explored in the memory test. In contrast, the rapamycin injected mice showed only a little preference for new xenobiotics, and the cognitive indices of the two groups were 0.68 ± 0.018 for the control group and 0.56 ± 0.023 for the rapamycin injected group (t ═ 4.067, P ═ 0.001), respectively. These results indicate that the rapamycin injected mice had severe impairment in new body recognition memory.
(2) Water maze experiment
The water maze experiment is an experiment that rodents can meet water and are afraid of the water and are placed in water to force the rodents to escape the water environment, so that the rodents learn to find the position of the water platform by using the relation between an environmental marker and a hidden platform, and the experiment is applied to the neurobiological mechanism discussion of learning and memory. The method comprises two parts of positioning navigation and space exploration test. The positioning navigation test is a platform which is trained for a plurality of times to learn and search a fixed position, and the platform is recognized by taking clues in the surrounding environment as reference space positions, and forms space reference memory, and the memory storage and extraction relate to the limbic system (hippocampus), striatum, basal forebrain, cerebellum and other brain areas. The space exploration test is mainly used for inspecting the memory of the mouse on the escape platform. If the experimental mouse in the water maze experiment can not find the platform position correctly, the space memory and cognition of the mouse are obstructed.
As shown in fig. 15: in the positioning navigation experiment of the water maze experiment, the escape latency of all mice is constantly reduced in the training process, which shows that the acquisition capacity of the space memory of the mice is enhanced along with the increase of the training days. Two groups of mice are similar in performance in the first two days of positioning navigation; and the escape latency of the rapamycin injected mice was significantly higher on the third, fourth and fifth days than the control mice (t and p values 3: t 2.211, p 0.04; 4: t 3.839, p 0.001; 5: t 5.109, p 0.000, respectively).
As shown in fig. 16: in terms of search distance, the rapamycin injection group showed a decreasing trend of "decrease-increase-decrease" fluctuation, which increased on day 3, while the control group showed a steadily decreasing trend, with a significant difference between the two groups on the fifth day of training (t 5.304, p < 0.05).
As shown in fig. 17: the mice injected with rapamycin crossed the original platform position significantly less frequently than the control mice (P < 0.05).
As shown in fig. 18: the speed difference of mouse swimming is not obvious in the whole course of the positioning navigation training.
As shown in fig. 19: the residence time of the rapamycin injected mice in the water maze space exploration experiment target quadrant is also significantly lower than that of the control group mice (P < 0.001).
As shown in fig. 20: after 5 days of the positioning navigation training, the platform is removed, a space exploration experiment is carried out, and a track graph of the platform searched by the mice is drawn, so that the reference memory capability of two groups of mice is compared, and whether the reference memory capability is influenced or not is judged. As can be seen from fig. 20: the control group of mice can quickly find the position of the platform according to a spatial cue, the search track is mostly in the quadrant where the original platform is located, and then in the adjacent quadrant of the quadrant where the original platform is located, and the control group of mice rarely cross the opposite side quadrant for searching. Whereas the rapamycin injected group of mice was mostly sought around the pool wall, rarely explored across diameters. The time percentage of rapamycin in the original platform quadrant in the rapamycin injected mice was significantly less than that in the control mice (mean + -SEM: 10.16 + -1.64, control: 31.71 + -1.56; t ═ 8.732, P < 0.001).
The sizes of the body types and the brain volumes of the filial generation mice of the over-injection rapamycin group and the control group are compared, and the social interaction, the movement and the cognitive function behaviors of the mice are tested, so that the intervention of the rapamycin medicament in the pregnancy period of the pregnant mice is proved, and the autism filial generation mouse model is obtained. Establishes a new reliable animal model for the related research of ASD and finds a new direction for the prevention and treatment of autism in the future.
The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.
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Claims (7)
1. a method for constructing an autism mouse model is characterized by comprising the following steps: the construction method comprises the steps of carrying out pregnancy intervention on a pregnant mouse by using a rapamycin medicament so as to obtain the autism offspring mouse model; wherein, for female healthy mice in the sexual maturity stage, the rapamycin solution is injected into the abdominal cavity at 3-7 mg/kg body weight from 11, 12 or 13 days of pregnancy until the pregnant mice give birth.
2. The method of constructing an autism mouse model of claim 1, wherein: for female healthy mice in the sexual maturity stage, a rapamycin solution was injected intraperitoneally at 5mg/kg body weight, starting at pregnancy 12 days, until delivery of pregnant mice.
3. The method of constructing an autism mouse model according to claim 1 or 2, wherein: the mice are C57BL/6 mice.
4. The method of constructing an autism mouse model of claim 1, wherein: rapamycin was dissolved in DMSO.
5. An autism mouse model characterized by: constructed according to the method of any one of claims 1 to 4.
6. Use of the mouse model of autism as defined in claim 5 for screening a therapeutic agent for treating autism.
7. Use of the mouse model of autism as defined in claim 5 for studying the pathogenesis of autism.
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