Mouse model for simulating neurodevelopment disorder

Abstract

The invention relates to the technical field of biological models, in particular to a mouse model for simulating neurodevelopmental disorder, which comprises the following steps: injecting a polyinosine-polycytidylic acid compound into a mouse during pregnancy to simulate maternal immune activation; separating the mother and the baby after the mouse is delivered, so as to obtain the mouse model for simulating the neurodevelopmental disorder. Through the mouse model, the common influence of multiple environmental factors on nervous system development can be more accurately researched, and the mouse model is particularly suitable for researching a mechanism of long-term psychological and behavioral effects and is beneficial to developing new intervention strategies and treatment methods.

Description

Technical Field

[0001] The invention relates to the technical field of biological models, and in particular to a mouse model for simulating neurodevelopmental disorders. Background Art

[0002] Current status of neurodevelopmental disorders (NDDs), which include a wide range of disorders such as autism spectrum disorder (ASD), intellectual development disorders (DID), attention deficit hyperactivity disorder (ADHD), epilepsy, schizophrenia (SZ), Rett syndrome, Down syndrome, cerebral palsy, fetal alcohol syndrome, childhood epilepsy, and many other rare genetic disorders that affect development, such as SLC6A1 disorders. NDDs affect >3% of children worldwide, constitute a serious health problem, and impose a huge economic burden on society and families. NDDs are characterized by the failure to achieve normal cognitive, emotional, and motor development. In general, NDDs are associated with disruptions in tightly coordinated events that lead to brain development. They have heterogeneous etiologies and result in impairments in cognition, communication, adaptive behavior, and psychomotor skills. ASD is the most common NDD, with 1 in 54 children. ASD is a group of developmental disorders whose main clinical manifestations are social communication dysfunction and repetitive stereotyped behaviors. Currently, a variety of genetic and environmental stressor rodent models have been used in the research community.

[0003] Over the past few decades, epidemiological studies have shown that MIA is an environmental risk factor for neuropsychiatric disorders such as SZ, ASD, and bipolar disorder (BD). Exposure to immune activation during the critical period of fetal brain development during pregnancy can lead to abnormal brain development in offspring and behavioral disorders in adulthood, including defects in social interaction and cognition. To study the effects of infection during pregnancy on brain function and behavioral phenotypes in adult offspring, specific MIA animal models based on the administration of immunogenic substances to pregnant females have been developed. The most commonly used method is to simulate MIA by treating with bacterial endotoxin lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) analog polyinosinic: polycytidylic acid (Poly(I:C)). It is now generally believed that most MIA do not directly infect the fetus through the placenta, but affect fetal neurodevelopment. Studies have shown that a single prenatal exposure to a "one-time" single dose (5-20 mg / kg) of Poly(I:C) or LPS is sufficient to impair hippocampal neurogenesis in adult offspring. However, epidemiological studies have shown that children exposed prenatally have the ability to recover from neurodevelopmental defects and remain healthy in adulthood. Exposure to MIA makes children susceptible to mental illness in adulthood, and offspring exposed to additional environmental "blows" such as stress or drug abuse are more likely to have behavioral abnormalities in adulthood. Therefore, it can be speculated that on the basis of the first blow based on MIA, giving offspring an additional second blow is more likely to induce neurobehavioral abnormalities in offspring.

[0004] Previous studies have shown that the presence of the mother plays an important role in the early development of newborn rodents. Newborn rodents maintain close contact with their mothers and can obtain various basic resources needed for survival, such as nutrition, body sensation, temperature, etc. These resources are essential for the formation of adult behavior, emotions and physiological responses. Clinical research evidence shows that the adverse effects of neglect, such as separation from the mother during the early development stage of children, may increase the risk of psychological disorders such as depression, ASD, psychosis and anxiety in adulthood. At present, the MS model in rodents is often used to study the effects of depression and early life stress on neurobiology, which may increase the risk of psychological disorders such as depression, ASD, psychosis and anxiety in adulthood. The MS model in rodents is often used to study the effects of depression and early life stress on neurobiology. There are two main models, MS15 (15 minutes of short MS) and MS180 (180 minutes of extended MS), from postnatal day 2 (P2) to P14, and even P21 when the pups are weaned. According to studies, long-term MS can induce increased depression-like and anxiety-like behaviors in offspring mice, and enhance learning and memory.

[0005] In existing studies, maternal immune activation (MIA) and maternal-fetal separation (MS) are often used alone to simulate neurodevelopmental disorders. However, individual simulation methods often fail to fully simulate the multifactorial complexity of human diseases. Therefore, there is a need to provide a more comprehensive simulation method to assist researchers in gaining a deeper understanding of the causes and development of neurodevelopmental disorders. Summary of the invention

[0006] The purpose of the present invention is to provide a mouse model that simulates neurodevelopmental disorders. Through the mouse model of the present invention, the combined effects of multiple environmental factors on the development of the nervous system can be more accurately studied. It is particularly suitable for studying the mechanisms of long-term psychological and behavioral effects, and is helpful for developing new intervention strategies and treatment methods.

[0007] The present invention solves the technical problem by adopting the following technical solutions: In a first aspect, the present invention provides a mouse model for simulating neurodevelopmental disorders, comprising: Step 1: Mice were injected with polyinosinic: polycytidylic acid (Poly(I:C)) during pregnancy to simulate maternal immune activation; Step 2: After the mouse is delivered, the mother and the baby are separated to obtain a mouse model simulating neurodevelopmental disorders.

[0008] The animal model of this experiment was also constructed based on the classic ASD model induced by MIA. The core symptom of ASD is social deficit. The three-box experiment proved that the model mice showed the core symptoms of ASD.

[0009] MIA (maternal immune activation) in the present invention was previously used to model ASD, but the modeling was unstable, and not all offspring born to pregnant mice with maternal immune activation showed autism. Injecting polyinosinic: polycytidylic acid (Poly(I:C)) into pregnant mice to cause maternal immune activation and induce autism-like symptoms in their offspring is a classic ASD modeling method. MS (maternal-infant separation) was previously used more for modeling depression and anxiety.

[0010] The model of MIA and MS double hit is more complicated than the modeling of single factors, and the double hit caused by the double factor modeling is more stable for the model of neurodevelopmental disorders. The elevated plus maze and three-box social ability test proved that the mouse model simulating neurodevelopmental disorders of the present invention showed neurodevelopmental disorder behaviors, such as anxiety, depression (elevated cross), and impaired social ability (three-box social ability test).

[0011] The elevated plus maze experiment showed that the model mice had anxious behavior, and the three-box experiment showed that the model mice had impaired social skills, indicating that the double blow of MIA+MS caused neurodevelopmental disorders in mice.

[0012] In some embodiments of the present invention, in step 1: mice are intraperitoneally injected with 10-40 mg / kg of polyinosinic-polycytidylic acid complexes within 5-20 days of pregnancy to simulate maternal immune activation.

[0013] In some embodiments of the present invention, in step 1: mice are intraperitoneally injected with 10-30 mg / kg of polyinosinic-polycytidylic acid complexes within 10-15 days of pregnancy to simulate maternal immune activation.

[0014] In some embodiments of the present invention, in step 2: the mother-infant separation is performed on the second day after the mouse is delivered, and the mother-infant separation is continued for 5-30 days, thereby obtaining a mouse model simulating neurodevelopmental disorders.

[0015] In some embodiments of the present invention, in step 2: the mother-infant separation is performed on the second day after the mouse is delivered, and the mother-infant separation is continued for 10 to 20 days, thereby obtaining a mouse model simulating neurodevelopmental disorders.

[0016] In some embodiments of the present invention, in step 2: the mother and the infant are separated for 1 to 8 hours at a random time period every day after the mouse is delivered, and the mother and the infant are separated for 10 to 20 days to obtain a mouse model simulating neurodevelopmental disorders.

[0017] In some embodiments of the present invention, in step 2: the mother and the infant are separated for 2 to 4 hours at a random time period every day after the mouse is delivered, and the mother and the infant are separated for 12 to 18 days to obtain a mouse model simulating neurodevelopmental disorders.

[0018] In some embodiments of the present invention, in step 1: within 12 to 13 days of pregnancy, mice are intraperitoneally injected with 18 to 22 mg / kg of polyinosinic-polycytidylic acid complexes to simulate maternal immune activation; In the step 2, the mother and the infant are separated for 2.5 to 3.5 hours at a random time period every day after the mouse is delivered, and the mother and the infant are separated for 13 to 15 days to obtain a mouse model simulating neurodevelopmental disorders.

[0019] The present invention includes the following beneficial effects: The mouse model of the present invention, through social experiments and maze experiments, shows that the neurodevelopment of the mouse model is affected, resulting in obvious social ability impairment and anxiety, indicating that the model is successful. In addition, the data of experimental group three is significantly more concentrated than that of the other two groups, indicating that the mouse model of the present invention is more stable.

[0020] The mouse model of the present invention can be used to more accurately study the joint effects of multiple environmental factors on the development of the nervous system. Specifically, the double blow of MIA (maternal immune activation-infection during pregnancy) and MS (maternal-infant separation-childhood adversity) reflects the joint effects of multiple environmental factors on the development of the nervous system. It is suitable for studying the mechanisms of long-term psychological and behavioral effects and helps to develop new intervention strategies and treatments.

[0021] The mouse model of the present invention can be used to study and develop the effects of drugs on treating neurodevelopmental disorders caused by complex factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Behavioral room for three-box social experiments.

[0023] Figure 2 This is a diagram of the elevated plus maze experimental apparatus.

[0024] Figure 3 This is a bar graph showing the number of times each group of mice entered different areas in the three-box behavioral experiment.

[0025] Figure 4 This is a comparison chart of the number of times mice in experimental group one and experimental group three entered different areas in the three-box behavioral experiment.

[0026] Figure 5 This is a comparison chart of the number of times mice in experimental group two and experimental group three entered different areas in the three-box behavioral experiment.

[0027] Figure 6 This is a bar graph of the time each group of mice spent in the open arm in the elevated plus maze experiment.

[0028] Figure 7 This is a comparison of the time spent in the open arm by mice in experimental group one and mice in experimental group three in the elevated plus maze experiment.

[0029] Figure 8 This is a comparison of the time spent in the open arm by mice in experimental group 2 and mice in experimental group 3 in the elevated plus maze experiment.

[0030] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0031] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. DETAILED DESCRIPTION

[0032] The present invention specification describes the specific implementation scheme in detail. Those skilled in the art should recognize that the following implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The following examples specifically illustrate the beneficial effects of the present invention.

[0033] 1. Experimental Grouping Control group (PBS): C57 mice, 7 cases. The mother mice were intraperitoneally injected with an equal volume of PBS (the administration volume of PolyI:C for mice of the same weight) at 12.5 days of pregnancy. After the offspring were weaned at 3 weeks of age, the mothers and offspring were raised separately. When the mice were 6-8 weeks old, the neurodevelopmental disorder (NDDs) model was verified through animal behavior experiments (three-box social test, elevated plus maze test).

[0034] Experimental group 1 (MIA): C57 mice, 7 cases. The mother mice were intraperitoneally injected with 20 mg / kg of polyinosinic: polycytidylic acid (Poly(I:C)) at 12.5 days of pregnancy. After the offspring were weaned at 3 weeks of age, the mothers and offspring were raised separately. When the mice were 6-8 weeks old, the neurodevelopmental disorders (NDDs) model was verified through animal behavior experiments (three-box social test, elevated plus maze test).

[0035] Experimental group 2 (MS): C57 mice, 7 cases, the mother mice were intraperitoneally injected with an equal volume of PBS (the administration volume of PolyI: C for mice of the same weight) at 12.5 days of pregnancy, and maternal separation (MS) began on the second day after the offspring were born, and continued for 14 days, 3 hours / day. After the offspring were weaned at 3 weeks of age, the mothers and offspring were raised separately, and the neurodevelopmental disorder (NDDs) model was verified by animal behavior experiments (three-box social test, elevated plus maze test) at 6-8 weeks of age.

[0036] Experimental group 3 (MIA+MS): C57 mice, 7 cases, 20 mg / kg of polyinosinic: polycytidylic acid (Poly(I:C)) was intraperitoneally injected into the mother mice at 12.5 days of pregnancy, and maternal separation (MS) was performed from the second day after the offspring were born, which lasted for 14 days, 3 hours / day. After the offspring were weaned at 3 weeks of age, the mothers and offspring were raised separately, and the neurodevelopmental disorder (NDDs) model was verified by animal behavior experiments (three-box social test, elevated plus maze test) at 6-8 weeks of age.

[0037] 2. Behavioral Experiment 1. Three-box social experiment: (1) Behavioral room for three-box social experiment The experiment used Figure 1 The 60×40×22 cm transparent box shown was used as a behavioral room. Two removable transparent boards were used to divide the interior of the transparent box into three chambers: S1 area, S2 area, and the middle area between S1 area and S2 area. Each of the two removable boards had a 5×8 cm sliding door. After removing the removable boards, the three chambers were interconnected, and the mice could move freely inside.

[0038] (2) Three-box social experiment stage During the experiment, a metal cage with restraint function was placed in each of the two chambers in the S1 area and the S2 area. The metal cage was used to house the unfamiliar mice. The experimental mice and the unfamiliar mice were transported to the behavioral room to familiarize themselves with the environment 30 minutes before the experiment. The control group, experimental group 1 mice, experimental group 2 mice, and experimental group 3 mice were subjected to the following two-stage experiment.

[0039] Phase 1: Adaptation to the environment. Place a metal cage at the same position in each of the two chambers in the S1 area and the S2 area. Put the same building blocks in the metal cage. Put the experimental mouse in the chamber in the middle area. Open the sliding door and let it move freely for 5 minutes. Phase 2: Social ability test, the first stranger mouse was placed in the metal cage in the chamber of the S1 area. The stranger mouse was a mouse of the same age and gender from a different litter than the experimental mouse, recorded as stranger 1, and the metal cage with building blocks was placed in the S2 area, recorded as stranger 2. Then the experimental mouse was released from the chamber of the middle area, the sliding door was opened, and it was allowed to move freely for 10 minutes and recorded, and then the experimental mouse was returned to the chamber of the middle area and the sliding door was closed.

[0040] The movement trajectory and distance of the experimental mice, the number of times they entered each chamber, and the exploration time of unfamiliar mice or metal cages with building blocks were recorded. During the test, the test environment was kept quiet and the light intensity was 30 Lux. After the experiment, the experimental mice and unfamiliar mice were put back into the breeding cages and the experimental equipment was cleaned with 75% ethanol.

[0041] (3) Experimental results Figure 3 This is a bar graph showing the number of times each group of mice entered different areas in the three-box behavioral experiment. Figure 4 This is a comparison chart of the number of times mice in experimental group one and experimental group three entered different areas in the three-box behavioral experiment. Figure 5This is a comparison of the number of times mice in experimental group 2 and experimental group 3 entered different areas in the three-box behavioral experiment. Note: The second stage of the three-box social experiment tests the social ability of mice. In the figure, * represents a statistical P value <0.05, ** represents a statistical P value <0.01, *** represents a statistical P value <0.001, and **** represents a statistical P value <0.0001. The smaller the P value, the more significant the difference).

[0042] The results are analyzed as follows: like Figure 3 As shown, in the control group, the number of times that normal mice entered the S1 area (cage with mice) during the social ability test phase was significantly higher than that of the S2 area (empty cage), with a P value of <0.001, indicating that mice were significantly more interested in the cage with mice (S1 area) than in the empty cage (S2 area), and the mice in the control group had normal social ability; in the experimental group 1 (MIA) mice, the number of times that they entered the S1 area (cage with mice) was higher than that of the S2 area (empty cage) during the social ability test phase, indicating that mice were more interested in the cage with mice (S1 area) than in the empty cage (S2 area), but The P value was <0.05, and the P value increased and the difference decreased compared with the control group, indicating that the mice in experimental group 1 (MIA) had a certain degree of impaired social ability compared with the mice in the control group; there was no significant difference in the number of times the mice in experimental group 2 (MS) and experimental group 3 (MIA+MS) entered the S1 area and S2 area during the social ability detection phase, indicating that the mice in experimental group 2 (MS) and experimental group 3 (MIA+MS) had no significant difference in interest in cages with mice (S1 area) and empty cages (S2 area), and had obvious social ability impairment.

[0043] like Figure 4 As shown in the figure, the comparison of the two model mice in the social ability detection stage of experimental group 1 (MIA) and experimental group 3 (MIA+MS). There was no significant difference in the number of times the mice in experimental group 3 (MIA+MS) entered the S1 area (with mouse cage) and the S2 area (empty cage), indicating that the mice in experimental group 3 (MIA+MS) had no significant difference in interest in the S1 area (with mouse cage) and the S2 area (empty cage), and their social ability was significantly worse than that of experimental group 1 (MIA). The data of experimental group 1 (MIA) were obviously more scattered, and the data of experimental group 3 (MIA+MS) were more concentrated, indicating that the modeling of social ability impairment of experimental group 3 (MIA+MS) mice was more obvious and stable than the single MIA modeling (such as experimental group 1).

[0044] like Figure 5As shown in the figure, the comparison of the two model mice in experimental group 2 and experimental group 3 (MIA+MS) during the social ability detection stage showed that there was no significant difference in the number of times the mice in experimental group 3 (MIA+MS) entered the S1 area (with mouse cage) and the S2 area (empty cage), indicating that there was no significant difference in the interest of the mice in experimental group 3 (MIA+MS) in the S1 area (with mouse cage) and the S2 area (empty cage). In addition, the number of times the experimental group 2 entered the S1 area was significantly more than that of the S2 area, that is, the social ability of the experimental group 3 (MIA+MS) was significantly worse than that of the experimental group 2 (MS). The data of the experimental group 2 (MS) was significantly more scattered, and the data of the experimental group 3 (MIA+MS) was more concentrated, indicating that the modeling of the social ability impairment of the mice in the experimental group 3 (MIA+MS) was more obvious and stable than the single MS modeling (such as experimental group 2).

[0045] 2. Elevated plus maze test (1). Elevated plus maze experimental apparatus The experimental device used Figure 2 The elevated plus maze shown consists of a middle connecting platform (10×10 cm), two open arms and two closed arms, the open arms and the closed arms are 30 cm long and 6 cm wide, with 15 cm high wall panels on both sides of the closed arms. The device is opaque gray in color and 50 cm above the ground.

[0046] (2) Elevated plus maze test procedure 30 min before the start of the experiment, the experimental mice were transported to the behavioral room to familiarize themselves with the environment.

[0047] During the experiment, mice were placed in the control group (PBS), experimental group 1 (MIA), experimental group 2 (MS) and experimental group 3 (MIA+MS) from the middle connecting platform with their backs to the experimenter (as long as the mice were placed in the center of the maze with their backs to the experimenter, the reasons for this were: to reduce interference, lower stress response, improve the repeatability of the experiment and promote exploratory behavior) and allowed to explore freely. The situations of the mice in the control group (PBS), experimental group 1 (MIA), experimental group 2 (MS) and experimental group 3 (MIA+MS) entering the open arm and the closed arm within 10 min were recorded respectively.

[0048] During the test, the environment was kept quiet and the light intensity was 30 Lux. After the experiment, the experimental device was cleaned with 75% ethanol.

[0049] (3) Experimental results Figure 6 This is a bar graph of the time each group of mice spent in the open arm in the elevated plus maze experiment. Figure 7 This is a comparison of the time spent in the open arm by mice in experimental group one and mice in experimental group three in the elevated plus maze experiment. Figure 8 This is a comparison of the time spent in the open arm of the mice in the experimental group 2 and the mice in the experimental group 3 in the elevated plus maze experiment. Description: The elevated plus maze experiment detects whether the animals have anxiety-like behaviors. The results are analyzed as follows (in the figure, * represents a statistical P value < 0.05, ** represents a statistical P value < 0.01, *** represents a statistical P value < 0.001, **** represents a statistical P value < 0.0001, and the smaller the P value, the more significant the difference).

[0050] like Figure 6 As shown, the time that mice in experimental group 1 (MIA), experimental group 2 (MS), and experimental group 3 (MIA+MS) spent on open arm activities was significantly lower than that in the control group (PBS), indicating that mice in experimental group 1 (MIA), experimental group 2 (MS), and experimental group 3 (MIA+MS) had anxiety-like behaviors.

[0051] like Figure 7 As shown, the time that mice in experimental group three (MIA+MS) spent in open arm activities was significantly lower than that in experimental group one (MIA), indicating that the anxiety-like behavior of mice in experimental group three (MIA+MS) was more obvious than that in experimental group one (MIA).

[0052] like Figure 8 As shown, the time that mice in experimental group three (MIA+MS) spent in open arm activities was significantly lower than that in experimental group two (MS), indicating that the anxiety-like behavior of mice in experimental group three (MIA+MS) was more obvious than that in experimental group two (MS).

[0053] from Figure 6-8 It can be seen that when MIA+MS was used together to model, the anxiety-like behavior was significantly lower than that of the single model, that is, MIA+MS had a synergistic effect, that is, MIA+MS was significantly better than the effect of MIA or MS used alone.

[0054] The differences between experimental group 3 (MIA+MS) and experimental group 1 (MIA), and experimental group 3 (MIA+MS) and experimental group 2 (MS) were compared. From the data dispersion in the figure, it can be seen that the data of experimental group 1 (MIA) and experimental group 2 (MS) are relatively scattered, indicating that the anxiety-like behavior model under this model is not stable enough. However, when experimental group 3 (MIA+MS) was modeled by MIA+MS, the data was very concentrated, indicating that the model is very stable and easier to use commercially.

[0055] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A mouse model for simulating neurodevelopmental disorders, characterized in that: include: Step 1: During the 5th to 20th day of pregnancy, mice were intraperitoneally injected with 10-40 mg / kg of polyinosinic-polycytidylic acid complex to simulate maternal immune activation; Step 2: After the mouse is born, the mother and the baby are separated for 1 to 8 hours at a random time period every day, and the mother-baby separation is continued for 10 to 20 days to obtain a mouse model simulating neurodevelopmental disorders.

2. A mouse model for simulating neurodevelopmental disorders according to claim 1, characterized in that: In the step 1, mice are intraperitoneally injected with 10-30 mg / kg of polyinosinic-polycytidylic acid complexes within 10-15 days of pregnancy to simulate maternal immune activation.

3. A mouse model for simulating neurodevelopmental disorders according to claim 1, characterized in that: In the step 2, the mother and the infant are separated for 2 to 4 hours at a random time period every day after the mouse is delivered, and the mother and the infant are separated for 12 to 18 days to obtain a mouse model simulating neurodevelopmental disorders.

4. A mouse model for simulating neurodevelopmental disorders according to claim 1 or 2, characterized in that: In the step 1, mice were intraperitoneally injected with 18-22 mg / kg of polyinosinic-polycytidylic acid complexes within 12-13 days of pregnancy to simulate maternal immune activation; In the step 2, the mother and the infant are separated for 2.5 to 3.5 hours at a random time period every day after the mouse is delivered, and the mother and the infant are separated for 13 to 15 days to obtain a mouse model simulating neurodevelopmental disorders.

Images