Establishment method and application of autism animal model

Abstract

The invention discloses a construction method of an autism animal model, which is characterized in that drug administration is carried out for three times for the first time, and drug administration can be carried out for one time at the most accurate time for 100% of all animals, so that the optimal drug administration window period of 12.5 days of fertilized embryo pregnancy of all animals can be accurately and effectively covered; and the side effects of abortion, stillbirth, even pregnant mouse death and the like caused by the VPA on the pregnant female mouse can be effectively reduced. By means of the method, the human autism occurrence process can be better simulated, and the obvious autism behavior can be shown. According to the method, the modeling efficiency is greatly improved, the experiment cost is reduced, side effects are reduced, the death rate and abortion rate of pregnant test animals and newborn test animals are remarkably reduced, the experiment period is greatly shortened, the modeling efficiency is improved, a large amount of manpower and material resources and other modeling costs can be saved, animal welfare can be protected, and the method is suitable for popularization and application. The medicine research and development and the pathogenesis are promoted, and human early male autism spectrum disorder can be assisted.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for establishing an autism animal model and its application. Background Art

[0002] Autism spectrum disorder (ASD) is a psychiatric disorder associated with early neurodevelopmental disorders. ASD was first reported by American child psychiatrist Leo Kanner in 1943. The typical core symptoms of ASD are mainly social interaction disorders, repetitive stereotyped behaviors, and limited interests or activities. Some ASD patients also have intellectual disabilities and epilepsy. However, the biggest problem of ASD is caused by a series of other complications, such as irritability, anxiety, aggression, obsessive-compulsive disorder, emotional instability, gastrointestinal problems, depression, and sleep disorders. The Centers for Disease Control and Prevention reported that the prevalence of ASD in children is as high as 1.7%, with a male-to-female ratio of about 4.5:1. Globally, the prevalence of ASD is about 1%, showing an increasing trend year by year. Currently, there are about 80 million ASD patients worldwide. Once ASD occurs, most of its symptoms will accompany lifelong. About one-third of ASD children cannot live independently and need lifelong care, which brings heavy mental and economic burdens to families and society. Therefore, the disease has become a major public health problem recognized by the world. The lifetime social cost of ASD due to social assistance and loss of labor force required by patients and their parents is an average of $1.4 million to $2.4 million per child in the United States and £9,000 to £1.5 million in the United Kingdom. The annual social cost of ASD in the United States is approximately $236 billion, and in the United Kingdom it is approximately $47.5 billion.

[0003] At present, the cause and pathogenesis of ASD are still unclear, and there is no effective treatment, which has caused a serious burden on families and society. The prevalence of ASD in my country has also been increasing year by year, and is currently as high as 1%. According to the 2019 "China ASD Education and Rehabilitation Industry Development Status Report III", there are currently more than 10 million people with ASD in my country, including more than 2 million children with ASD aged 0-14, and the number is increasing at a rate of nearly 200,000 per year. At present, due to the unknown cause of ASD, the extremely high disability rate and the inability to cure it, lifelong intervention is still required. The prevalence of ASD has been increasing year by year, which has increasingly attracted widespread attention and concern from the entire society and the medical community. Moreover, its treatment has always been a global problem and has increasingly become a hot spot and difficulty in today's biomedical research. The prevalence of ASD in children around the world has made ASD a social problem that humans urgently need to solve today.

[0004] Although using ASD patients as research subjects is the most direct research method, due to ethical restrictions and the safety of stem cell therapy that has yet to be evaluated, stem cells cannot currently be widely used in the clinical treatment of ASD patients. Basic research on stem cell therapy can only be carried out on ASD animal models. This shows that constructing an animal model that can well simulate the various phenotypes of ASD and, on this basis, conducting in-depth research on stem cell therapy for ASD and its related mechanisms is the key to finding an effective way to prevent and treat ASD.

[0005] Currently, a variety of ASD animal models have been successfully established, among which the VPA-induced rat model is a classic animal model that can accurately simulate autism spectrum disorders. Rodents themselves have the advantages of low price, short gestation period, and large litter size, and are very similar to humans in electrophysiology, neuroanatomy, and genetics, which facilitates the study of related disease mechanisms. However, in the traditional VPA modeling method, pregnant mice have high mortality and miscarriage rates after administration, and the success rate of modeling is low. Therefore, it is of great significance to provide an improved method for establishing an animal model. Summary of the invention

[0006] The first aspect of the present invention provides a method for establishing an autism animal model, comprising administering sodium valproate to the test animal three times when the test animal is 11-14 days pregnant, wherein the dose of sodium valproate administered to the test animal each time is less than 600 mg·kg -1 , the test animals are rodents in a pregnant state.

[0007] High doses of sodium valproate have strong toxic side effects. The animal model establishment method of a single injection of high doses of sodium valproate in the prior art found that the parental test animals (i.e., pregnant rodents) had high mortality and abortion rates after administration, and the modeling success rate was extremely low, resulting in a large waste of manpower, material resources and other costs. -1The improved modeling method of sodium valproate administered to test animals in three doses has less side effects on the test animals, lower mortality and abortion rates, can improve modeling efficiency, and greatly save modeling costs. The establishment method in the present invention can significantly reduce various adverse reactions after sodium valproate administration to test animals. Although the test animals also show adverse reactions such as movement disorders, slow movements, and slow reactions to a certain extent after each administration of sodium valproate, such adverse reaction symptoms appear later and last for a shorter period of time, and adverse reactions such as body stiffness are very mild, and no phenomena such as body paralysis, inability to walk, and difficulty breathing are observed within 30 minutes, which significantly reduces the pain of animals and is more in line with the welfare and ethical requirements of experimental animals. Moreover, sodium valproate is administered in three doses, which will make the drug stay in the body of the test animals longer and more stably. Compared with traditional methods, this will cause more serious damage to the brain development of future test animals. Moreover, it is mainly by using a lower dose of sodium valproate in three doses with a certain interval between each dose. Compared with the one-time administration of a very large dose of sodium valproate, the effect on the test animals is milder. Moreover, this method of administration is closer to the pathological process of ASD in children induced by long-term and multiple intake of sodium valproate in clinical practice than a single dose.

[0008] In some embodiments, the second administration of sodium valproate to the test animal is when the test animal is 12.5 days pregnant, the first administration of sodium valproate to the test animal is 0.5 days or more from the second administration of sodium valproate to the test animal, and the third administration of sodium valproate to the test animal is 0.5 days or more from the second administration of sodium valproate to the test animal.

[0009] The critical window period for constructing an autism animal model by using sodium valproate for exposure during pregnancy is the time point of neural tube closure around the 12.5th day of pregnancy. This time point is considered to be a particularly vulnerable critical time window period that may cause autism. The use of sodium valproate can cause the development of these neurons and their corresponding cranial nerve nuclei to be disordered, such as inhibiting excessive proliferation of neurons, inhibiting the development and migration of neural crest cells, thereby leading to low differentiation, etc. In the present invention, sodium valproate is given three times around the 12.5th day of pregnancy of the test animal, and 3 sodium valproate absorption peaks higher than 10 times the maximum dose will appear around the critical time window period of 12.5d for the development of the nervous system of the offspring animal, which will undoubtedly cause the brain development of the offspring animal to be hit by 3 times the long time, which will undoubtedly make the development of the nervous system of the offspring animal more seriously damaged. The administration time point selected in the present invention can better induce the parental test animal to produce offspring animals (i.e., autism animal models) with significant social disorders, narrow interests, and repetitive stereotyped behaviors related to autism spectrum disorders.

[0010] In some embodiments, the first administration of sodium valproate to the test animal is at day 12 of gestation, the second administration of sodium valproate to the test animal is at day 12.5 of gestation, and the third administration of sodium valproate to the test animal is at day 13 of gestation.

[0011] In some embodiments, the first administration of sodium valproate to the test animal is at day 11 of gestation, the second administration of sodium valproate to the test animal is at day 12.5 of gestation, and the third administration of sodium valproate to the test animal is at day 13 of gestation.

[0012] In some embodiments, the first administration of sodium valproate to the test animal is at 11.5 days of gestation, the second administration of sodium valproate to the test animal is at 12.5 days of gestation, and the third administration of sodium valproate to the test animal is at 13.5 days of gestation.

[0013] In the present invention, different doses of sodium valproate are administered three times around 12.5 days of pregnancy, which can ensure that the difficulty in accurately and effectively administering sodium valproate exposure-induced obtaining an autism animal model during the dosing window period of 12.5 days of pregnancy due to the inability to accurately determine the conception time of the parent test animals during the cohabitation period and the inability to accurately calculate the embryo fertilization time can be reduced, resulting in a low efficiency of obtaining an autism animal model by one-time administration of sodium valproate exposure-induced obtaining, and only a small number of offspring animals born to pregnant test animals can effectively simulate the core symptoms of autism spectrum disorders.

[0014] In some embodiments, the subject animal is a rat and / or a mouse.

[0015] By adopting the construction method of the present invention, both C57BL / 6 mice and SD rats can efficiently obtain autism animal models. Taking into account the maternal pregnancy and delivery conditions, modeling costs, and apparent validity of the model, SD rats are more preferred.

[0016] In some embodiments, when the test animal is a rat, the dose of sodium valproate administered to the test animal each time is 300-500 mg·kg -1 .

[0017] In some embodiments, when the test animal is a mouse, the dose of sodium valproate administered to the test animal each time is 200-400 mg·kg -1 .

[0018] In some embodiments, the method comprises administering sodium valproate to rats in three divided doses when the rats are 11-14 days pregnant, wherein:

[0019] At 11 days of gestation, sodium valproate was given at a dose of 300 mg kg-1;

[0020] At 12.5 days of gestation, sodium valproate was given at a dose of 450 mg kg-1;

[0021] At 13 days of pregnancy, sodium valproate was given at a dose of 300 mg kg-1.

[0022] In some embodiments, the method comprises administering sodium valproate to rats in three divided doses when the rats are 11-14 days pregnant, wherein:

[0023] At 11.5 days of gestation, sodium valproate was given at a dose of 400 mg kg-1;

[0024] At 12.5 days of gestation, sodium valproate was given at a dose of 450 mg kg-1;

[0025] At 13.5 days of gestation, sodium valproate was given at a dose of 400 mg kg-1.

[0026] In some embodiments, the method comprises administering sodium valproate to mice three times when the mice are 11-14 days pregnant, wherein:

[0027] At 12 days of gestation, sodium valproate was given at a dose of 200 mg kg-1;

[0028] At 12.5 days of gestation, sodium valproate was given at a dose of 300 mg kg-1;

[0029] At 13 days of pregnancy, sodium valproate was given at a dose of 200 mg kg-1.

[0030] In some embodiments, the method comprises administering sodium valproate to mice three times when the mice are 11-14 days pregnant, wherein:

[0031] At 11.5 days of gestation, sodium valproate was given at a dose of 300 mg kg-1;

[0032] At 12.5 days of gestation, sodium valproate was given at a dose of 400 mg kg-1;

[0033] At 13.5 days of gestation, sodium valproate was given at a dose of 300 mg kg-1.

[0034] In some embodiments, each administration of sodium valproate is administered once via intraperitoneal injection.

[0035] The second aspect of the present invention provides an autism animal model obtained by the establishment method of the first aspect.

[0036] The third aspect of the present invention provides the use of the establishment method of the first aspect or the autism animal model of the second aspect in any of the following:

[0037] Study the causes and / or pathogenesis of autism;

[0038] Prepare products for studying the causes and / or pathogenesis of autism;

[0039] Screening for drugs to prevent and / or treat autism;

[0040] Preparation of products for screening drugs for the prevention and / or treatment of autism;

[0041] Preparation of products for studying the mechanism of action and / or therapeutic effect of autism treatment methods, such as studying the mechanism of action and / or therapeutic effect of stem cell therapy for autism.

[0042] The beneficial effects of the present invention are:

[0043] The method for constructing an autism animal model provided by the present invention can better simulate the process of human autism and can show obvious autism-like behaviors. The low modeling efficiency of the traditional VPA exposure modeling method by intraperitoneal injection may be mainly because the conception time of the female mouse during the cohabitation period is sometimes advanced and sometimes postponed, resulting in the difficulty in accurately calculating the administration window period of 12.5 days of pregnancy. The present invention is the first to propose three administrations, which truly achieves that 100% of all animals can be given a drug at the most accurate time, which not only ensures that the best administration window period of 12.5 days of fertilization embryo pregnancy of all animals can be accurately and effectively covered, but also effectively reduces the side effects of miscarriage and stillbirth caused by VPA to pregnant female mice and even the death of pregnant mice. The present invention greatly improves the modeling efficiency, reduces the experimental cost, reduces the side effects, significantly reduces the mortality rate and abortion rate of pregnant test animals and newborn test animals, greatly shortens the experimental cycle, improves the modeling efficiency, can save a lot of modeling costs such as manpower and material resources, and is conducive to protecting animal welfare, promoting drug development and pathogenesis research, and can help humans overcome autism spectrum disorders as soon as possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 These are the physiological development indicators of male offspring born in C57BL / 6 mice. A: The time of incisor eruption of male offspring born in C57BL / 6 mice; B: The age at which eye opening of male offspring born in C57BL / 6 mice reaches positive; C: The age at which abdominal hair growth of male offspring born in C57BL / 6 mice reaches positive. *P<0.05, **P<0.01, compared with the normal control group.

[0045] Figure 2These are neurodevelopmental indicators of male offspring born from C57BL / 6 mice. A: The time when the plane righting reflex behavior of male offspring born from C57BL / 6 mice reaches positive; 3B: The time when the aerial righting reflex behavior of male offspring born from C57BL / 6 mice reaches positive; C: The time when the cliff avoidance reflex behavior of male offspring born from C57BL / 6 mice reaches positive; *P<0.05, **P<0.01, compared with the normal control group.

[0046] Figure 3 The results of the observation of motor coordination and muscle tension of male offspring born C57BL / 6 mice. A: The time when the straight line crawling behavior of male offspring born C57BL / 6 mice reaches positive; B: The time when the grabbing behavior of male offspring born C57BL / 6 mice reaches positive.

[0047] Figure 4 It is the time when the auditory startle reflex of male mice born in C57BL / 6 reaches positive.

[0048] Figure 5 The results of the open field test of male pups born C57BL / 6. A: The number of times male pups born C57BL / 6 crossed the central grid; B: The vertical score of male pups born C57BL / 6 in the open field; C: The representative trajectory of male pups in the control group in the open field test; D: The representative trajectory of male pups in the two improved groups in the open field test.

[0049] Figure 6 This is a three-box social behavior experiment of male offspring born in C57BL / 6 mice. A: The first stage of social interaction experiment of male offspring born in C57BL / 6; B: The second stage of social preference experiment of male offspring born in C57BL / 6. *P<0.05, **P<0.01, compared with the normal control group; C: The representative trajectory of male offspring in the control group in the three-box social experiment; D: The representative trajectory of male offspring in the modified group 2 in the three-box social experiment.

[0050] Figure 7 The repetitive stereotyped behaviors of male offspring born to C57BL / 6 mice. A: self-grooming time; B: number of buried marbles.

[0051] Figure 8 These are physiological development indicators of male offspring born from SD rats. A: The time of incisor eruption of male offspring born from SD rats; B: The age at which eye opening of male offspring born from SD rats reaches a positive age; C: The age at which abdominal hair growth of male offspring born from SD rats reaches a positive age. *P<0.05, **P<0.01, compared with the normal control group.

[0052] Fig. 9These are neurodevelopmental indicators of male offspring born from SD rats. A: The time when the plane righting reflex behavior of male offspring born from SD rats reaches positive; B: The time when the aerial righting reflex behavior of male offspring born from SD rats reaches positive; C: The time when the cliff avoidance reflex behavior of male offspring born from SD rats reaches positive; *P<0.05, **P<0.01, compared with the normal control group.

[0053] Fig.10 The results of the observation of motor coordination ability and muscle tension of male offspring born from SD rats. A: The time when the straight line crawling behavior of male offspring born from SD rats reaches positive; B: The time when the pole grasping behavior of male offspring born from SD rats reaches positive.

[0054] Fig.11 It is the time when the auditory startle reflex of male offspring of SD rats becomes positive.

[0055] Fig.12 The results of the open field test of male offspring born in SD rats. A: The number of times male offspring born in SD rats crossed the central grid; B: The vertical score of male offspring born in SD rats in the open field; C: The representative trajectory of male offspring in the control group in the open field test; D: The representative trajectory of male offspring in the two improved groups in the open field test.

[0056] Fig.13 This is a three-box social behavior experiment of male offspring born from SD rats. A: The first stage of social interaction experiment of male offspring born from SD rats; B: The second stage of social preference experiment of male offspring born from SD rats. *P<0.05, **P<0.01, compared with the normal control group; C: The representative trajectory of male offspring in the control group in the three-box social experiment; D: The representative trajectory of male offspring in the modified group 2 in the three-box social experiment.

[0057] Fig.14 The repetitive stereotyped behaviors of male offspring of SD rats. A: self-grooming time; B: number of buried marbles. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below by specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0059] The experimental animals and some materials used in the examples of the present invention are as follows:

[0060] Adult C57BL / 6 mice, 24 female mice, 12 male mice, body weight 20-30g; adult SD rats, 24 female mice, 12 male mice, body weight 250-300g, purchased from Zhuhai Baishitong Biotechnology Co., Ltd. (Experimental Animal Production License No.: SCKK (Guangdong) 2020-0051), C57BL / 6 mouse animal qualification certificate No.: NO.44822700030567; SD rat animal qualification certificate No.: NO.44822700030566. They were kept in the SPF animal room of Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Experimental Animal Use License No.: SYXK (Guangdong) 2022-0063). Drinking water and food were given according to normal standards, and standard rat breeding feed and drinking water were available to mice at will. The constant temperature was maintained at 22±2℃, the relative humidity was 50%-60%, and the light was alternating day / night for 12h / 12h.

[0061] Sodium valproate (VPA) (Sigma, St. Louis, MO); observation panel for plane correction (20 cm × 20 cm × 2 cm), metal block for auditory startle reflex (1 cm 3 ), metal plate (35cm×30cm×0.5cm); three-box social experimental apparatus (mice: 120cm×20cm×22cm; rats: 120cm×45cm×40cm) and open field box (the size of the mouse activity box: 50cm×50cm×30cm, the size of the rat activity box: 100cm×100cm×45cm, the bottom of the box was divided into 25 equally divided small squares with a black marker, of which the 16 squares close to the edge were recorded as surrounding squares, and the 9 squares in the center were recorded as central squares).

[0062] Example 1 Preparation of VPA-induced ASD animal model - C57BL / 6 mice

[0063] Male and female adult C57BL / 6 mice and SD rats were caged together at 17:00 in the afternoon in the SPF animal room of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. The vagina of the female mice was checked at 9:00 on the second day after caged together. If a vaginal plug was found, it was considered to be fertilized successfully (i.e., pregnant mice). The day when the vaginal plug was found was recorded as the first day of pregnancy.

[0064] C57BL / 6 mice were randomly divided into 4 groups according to the VPA administration method:

[0065] (1) Traditional model group:

[0066] C57BL / 6 mice were given VPA 600 mg kg intraperitoneally at 12.5 days of gestation. -1 .

[0067] (2) Improved model group 1:

[0068] C57BL / 6 mice were given VPA 200 mg kg by intraperitoneal injection at 12 days of gestation. -1 ;

[0069] At 12.5 days of gestation, VPA 300 mg kg was given by intraperitoneal injection once. -1 ;

[0070] At 13 days of gestation, VPA 200 mg kg was given once by intraperitoneal injection. -1 .

[0071] (3) Improved model group 2:

[0072] C57BL / 6 mice were given VPA 300 mg kg by intraperitoneal injection at 11.5 days of gestation. -1 ;

[0073] At 12.5 days of gestation, VPA 400 mg kg was given by intraperitoneal injection once. -1 ;

[0074] At 13.5 days of gestation, VPA 300 mg kg was given by intraperitoneal injection once. -1 .

[0075] (4) Normal saline control group:

[0076] C57BL / 6 mice were given 600 mg kg of normal saline by intraperitoneal injection at 12.5 days of gestation. -1 .

[0077] After group drug administration:

[0078] ①Observe the adverse reactions, mortality and abortion rate of C57BL / 6 pregnant mice in each group after administration of sodium valproate or normal saline;

[0079] ②Observe the morphology, growth and development of the newborn mice in each group;

[0080] ③ Observe the social behavior of each group of newborn mice, for example: including three-box social behavior experiments, youth play experiments and open field experiments, etc., various indicators reflecting autism-like behaviors of the mice.

[0081] The specific observation methods are as follows:

[0082] 1 Observation and statistical analysis of the birth rate of pregnant rats and the rate of appearance deformity of offspring in each group

[0083] ① The birth rate of pregnant mice: the number of pregnant mice in each group and the number of C57 mice and SD rats that eventually gave birth (i.e. the number of pregnant mice that gave birth) were counted respectively.

[0084] The formula for calculating the birth rate (%) of pregnant mice is: number of pregnant mice delivered / number of pregnant mice in pregnancy × 100%.

[0085] ② The rate of external deformities in born mice: observe the external development of the skull and limbs of the mice (including male and female mice) in each group on the day of birth; count the number of external deformities (i.e. short limbs, unclear eyeballs and auricles) in each group (except for tail deformities) (Fig.

[0086] The calculation formula for the rate of external deformity of offspring (%) is: number of deformed offspring / total number of offspring born × 100%.

[0087] 2 Statistical analysis of adverse reactions and deaths in pregnant mice in each group after administration

[0088] After the drug was administered via intraperitoneal injection, the pregnant mice were gently returned to the cages to observe the adverse reactions of the mice in each group within 30 minutes after the intraperitoneal injection and the death of the mice in each group within 6 hours.

[0089] 3 Statistical analysis of abortion rate of pregnant mice in each group after drug administration

[0090] ① At 9:00 a.m. the next day after cage-warming, check the animals in each group for vaginal plugs. If vaginal plugs are found,

[0091] ② At 12.5 days of pregnancy, the body weight increases significantly and the abdomen bulges significantly;

[0092] ③There is a sudden decrease in body weight during the 19th to 20th day of pregnancy.

[0093] If the pregnant mother mouse can meet the above three detection criteria at the same time, it can be judged as miscarriage.

[0094] The statistical calculation formula for abortion rate is: number of aborted pregnant mice in each group / total number of pregnant mice in each group × 100%.

[0095] 4 Observation and statistical analysis of physiological development indicators of male offspring born in each group

[0096] ① 4-day survival rate of pups: The number of surviving male pups in each group was observed and counted on the 4th day after birth. The 4-day survival rate (%) was calculated as follows: number of 4-day-old live male pups / number of live male pups at birth × 100%.

[0097] The body weight and tail length of male pups in each group were measured on the 7th, 14th, 21st and 28th days after birth.

[0098] ② Method for measuring the body weight of young mice: Place the young mice on the scale and let them rest for 2 seconds before reading the numbers. Measure each animal 3 times and calculate the average. The unit of body weight is milligrams (mg). In order to maintain the consistency of the sample quantity, necessary killing was carried out.

[0099] ③Method for measuring the tail length of the pup: Starting from the base of the pup's tail, measure the length of the pup's tail when it is in a straight position. Measure each animal three times and calculate the average. The unit of tail length is centimeters (cm).

[0100] ④ Tail appearance deformity rate: The tail deformity (i.e., tail bending) of adult male mice in each group was observed on the 30th day after birth.

[0101] The calculation formula for the tail appearance deformity rate (%) was: the number of male offspring with bent tails after adulthood / the total number of male offspring after adulthood × 100%.

[0102] The male offspring were observed for the age at which incisor eruption, eye opening and fur development reached a positive age.

[0103] ⑤ The eruption of incisors reaches the positive judgment standard: white spots appear on both sides of the lower incisors of the pups visible to the naked eye, and a tingling sensation after touching with the hand is positive; starting from the 7th day after birth, each pup is tested one by one, and the age of all the pups born to pregnant mice in each group that meet the standard is recorded separately.

[0104] ⑥ Eye opening reaches the positive judgment standard: the eyelids of the pups are tightly closed when they are just born, and they gradually open their eyes after 8 days of age. The pups are considered positive if the degree of opening of both eyes is ≥ 1 / 2 of the eye sockets. The test starts from the 8th day after birth, and the age of all the pups born to pregnant mice in each group that meet the standard is recorded.

[0105] ⑦ Abdominal hair growth reaches the positive judgment standard: when the abdomen changes from pink to white (SD rats) or black (C57 mice) with hair, it is considered positive. Test each mouse one by one from the 10th day after birth, and record the age of all the pups born by each group of pregnant mice when they reach the standard.

[0106] Observation and statistical analysis of the development of the nervous system of male offspring in each group

[0107] The neuroreflex development indicators of male mice in each group were observed, including surfacing righting reflex, cliff avoidance reflex, air righting reflex, number of days when pivoting behavior reached positive, etc.

[0108] ① The plane righting reflex reaches the positive judgment standard: Place the young mouse on a wooden flat board and fix it with both hands

[0109] Maintain the supine position for 3 seconds, then gently release the hand, and start timing at the same time. The first positive righting reflex is defined as the ability of the pup to turn over to the prone position within 10 seconds and touch the flat surface with four paws flush. All pups were examined and recorded every day, and the age of all pups born to pregnant mice in each group that met the standard was recorded separately.

[0110] ② Cliff avoidance reflex reaches the positive judgment standard: Place the mouse on the edge of a 30 cm high platform with its neck exposed.

[0111] If the mice can turn around or retreat within 10 seconds, it is considered positive. The test starts from the second day after birth, and the age of all the pups born by pregnant mice in each group that meet the standard is recorded.

[0112] ③ The air righting reflex reaches the positive judgment standard: the tested mouse is fixed in the supine position in the air for 3 seconds, then gently let go of the hand to let it fall naturally onto the soft towel cushion prepared in advance, and the height of the young mouse from the cushion is 30 cm. If the tested mouse can land with a normal abdomen and limbs facing down for 3 consecutive times, the air righting reflex is considered positive, if not, it is negative. Start testing one by one from the 7th day after birth, and record the age of all the pups born by pregnant mice in each group that meet the standard.

[0113] ④ The positive judgment standard for rotation is reached: if the offspring can rotate clockwise or counterclockwise in 3 minutes without moving forward or backward, it can be judged as positive. Test each offspring one by one from the 2nd day after birth, and record the age of all offspring born by pregnant mice in each group when they meet the standard.

[0114] 6. Observation of motor coordination ability and muscle tension of male offspring in each group: Observe the number of days when the male offspring's bar holding test and crawling behavior reach positive, which reflects their motor coordination ability and muscle tension.

[0115] ① The grasping pole meets the positive judgment standard: the front legs of the pups are placed on a horizontal pole about 25 cm high. If the pups can hold the horizontal pole and hang for ≥ 2 seconds, it can be judged as positive. Starting from the 4th day after birth, the tests are conducted one by one, and the ages of all the pups born to pregnant mice in each group that meet the standard are recorded separately.

[0116] ② Crawling meets the positive judgment standard: If the puppies can crawl forward 3 cm in a straight line on all fours without leaving the platform, it can be judged as positive. Starting from 2 days after birth, each pup will be tested one by one, and the age of all the pups born to pregnant mice in each group that meet the standard will be recorded separately.

[0117] 7 Auditory startle reflex: The number of days on which the auditory startle reflex of male mice in each group reached a positive level was observed.

[0118] Auditory startle reflex reaches the positive judgment standard: place a metal plate parallel to the pups, 15 cm above the metal plate, keep the surroundings quiet, drop a metal block vertically to hit the metal plate below, and make a sound. If the pups curl up or tremble after hearing the sound, it can be judged as positive. Test each pup from 2 days after birth, and record the age of all pups born to pregnant mice in each group that meet the standard.

[0119] 8Behavioral testing

[0120] The pups in each group were subjected to behavioral testing for one week starting on the 28th day after birth, and cardiac perfusion sampling was started on the 35th day after birth.

[0121] 8.1 Social interaction behavior experiment

[0122] (1) Early adaptation stage:

[0123] Gently place the mice to be tested into the experimental apparatus (material: acrylic glass plate, mouse activity box size: 50cm×50cm×30cm, rat activity box size: 100cm×100cm×45cm), and allow them to move freely in the activity box for 10 minutes.

[0124] (2) Formal testing phase:

[0125] Take out the test mouse that has just adapted, and put in the test mouse and the interactive mouse (a mouse of the same strain with the same gender and similar age that has not been raised in the same cage as the test mouse) at the same time, and record the time when the test mouse and the interactive mouse have aggressive and non-aggressive behaviors in the next 10 minutes.

[0126] Aggressive behaviors include: the tested mouse actively scratches or bites the interactive mouse with its mouth; the tested mouse and the interactive mouse perform unfriendly behaviors such as standing and "glaring" at each other.

[0127] Non-aggressive behaviors include friendly behaviors such as the tested mouse actively chasing, following, approaching the interactive mouse and having close physical contact.

[0128] After the test, take out the tested mice and the interactive mice. After each round of the experiment, clean up the feces, urine and other excrement in time. Wipe the experimental device with 75% alcohol, dry it, remove the odor left by the previous animal as much as possible, and then replace the next mouse for behavioral testing.

[0129] 8.2 Youth Play Experiment

[0130] (1) Early adaptation stage:

[0131] The test mouse was gently placed into the experimental apparatus (material: acrylic glass plate, mouse activity box size: 50cm×50cm×30cm, rat activity box size: 100cm×100cm×45cm), and allowed to move freely in the activity box for 10 minutes.

[0132] (2) Formal testing phase:

[0133] Take out the test mouse that has just been adapted, and put the test mouse and the interaction mouse (same as before) in at the same time. Record the time of a series of interactive behaviors (including: social behavior, self-grooming and digging behavior) between the test mouse and the interaction mouse in the next 10 minutes.

[0134] Social behaviors include: the tested mouse actively chases, follows, approaches, and actively puts its forelimbs on the interactive mouse; the tested mouse actively strokes the interactive mouse's fur; the tested mouse actively makes nose-to-nose or nose-to-body contact with the interactive mouse, etc.

[0135] Self-grooming behavior: The test rats perform repetitive actions of grooming any part of their body with their tongues.

[0136] Digging behavior: The test rats dig with their forelimbs.

[0137] After the test, take out the tested mice and the interactive mice. After each round of the experiment, clean up the feces, urine and other excrement in time. Wipe the experimental device with 75% alcohol, dry it, and remove the odor left by the previous animal as much as possible. Then replace the next mouse for behavioral testing.

[0138] 8.3 Open field experiment

[0139] The open field test is a classic behavioral experimental method to evaluate the degree of exploration, autonomous action, and anxiety of experimental animals in a completely new environment. When rodents are in an open place, they have an exploratory nature towards the center of the open place, but at the same time they are afraid of the open environment. The open field test places the experimental rodents in an unfamiliar open environment to prevent them from escaping. The contradiction between the fear and curiosity of the experimental rodents towards the open place will cause them to feel anxious, so the open field test can be used to evaluate the anxiety of animals. The open field behavior analysis mainly reflects the animal's exploration habits of the new environment and the accompanying emotional changes. It can well assess the animal's emotional level and exploratory behavior in an open and unfamiliar environment, and can be used to test the "depressed" or "excited" state of the animal's central nervous system. The number of standing times is a conventional indicator in the analysis of the open field test, and is often used to reflect the mouse's ability to explore the environment. A large number of standing times indicates a strong ability to explore.

[0140] (1) Early adaptation stage:

[0141] The test mouse was gently placed into the experimental apparatus (material: acrylic glass plate, mouse activity box size: 50cm×50cm×30cm, rat activity box size: 100cm×100cm×45cm), and allowed to move freely in the activity box for 10 minutes.

[0142] (2) Formal testing phase:

[0143] The number of times the tested mouse crossed the central grid, the number of times it crossed the surrounding grids, the vertical score (number of standing times and wall climbing times) and the urination and defecation of the tested mouse during the test phase were recorded in the next 10 minutes.

[0144] The experimental device is divided into 16 grids in total, the 4 grids in the middle are the central grids, and the 12 grids around are the peripheral grids. The criterion for crossing the grid is: all four limbs of the test mouse cross the grid edge.

[0145] Vertical score (standing and wall climbing criteria): Both front limbs are off the ground and maintained for 2 seconds or more.

[0146] After the test, take out the tested mice and the interactive mice. After each round of the experiment, clean up the feces, urine and other excrement in time. Wipe the experimental device with 75% alcohol, dry it, and remove the odor left by the previous animal as much as possible. Then replace the next mouse for behavioral testing.

[0147] 8.4 Three-Box Social Behavior Experiment

[0148] The three-box social test is one of the most commonly used methods to assess the social tendencies and social novelty preferences of rodents. It can provide multiple measures of the social abilities of the tested mice.

[0149] (1) Early adaptation stage:

[0150] The three-box social device consists of a three-chamber transparent plexiglass box, which is divided into a middle chamber, a left chamber and a right chamber. There is an opening between the left and right chambers and the central chamber. Material: acrylic glass, (mice: 120cm×20cm×22cm; rats: 120cm×45cm×40cm). Before the experiment, the test mice need to be placed in the three-box experimental device and allowed to freely explore in the three interconnected chambers for a period of time to adapt to the experimental environment. In the present invention, the test mice are moved into the test room for 2 hours every day one week before the experiment to make them familiar with the experimenter and the room. Starting from 3 days before the formal test, the test mice are placed in the middle chamber of the plexiglass box every day, and the channels on both sides are temporarily closed with glass plates. After 3 minutes, the channels are opened to allow the mice to freely explore the chambers on both sides for 3 minutes, so that the test mice can fully adapt to the three-chamber experimental device. On the day of the experiment, empty cages are placed in the chambers on both sides, and the test animals are allowed to freely explore the three chambers for 5 minutes again. Then the test officially begins.

[0151] (2) The formal experiment is divided into two stages:

[0152] ① Social test:

[0153] A male stranger mouse (Stranger 1) of the same age and different litter (same sex, similar age and same strain as the test mouse and not previously caged together) was placed in an inverted metal coil in one chamber, which was labeled as stranger 1 cage. In the other chamber, only an inverted transparent metal coil was placed, which was labeled as object cage. The test mouse was placed in an empty central cage, and the activity of the test mouse and the duration of its olfactory contact with stranger 1 or the object were observed for 10 minutes.

[0154] ②Social preference test:

[0155] After the first stage of social testing, another stranger mouse 2 (Stranger 2) of the same age, gender and different litter was placed in the empty inverted metal coil and observed for another 10 minutes. The camera system and computer software recorded the time the tested mouse stayed in each side chamber and the time it communicated with Stranger 1 and Stranger 2 through sniffing. After the test, the tested mouse and the stranger mice Stranger 1 and Stranger 2 in the inverted metal coils on both sides were taken out in turn and put back into different cages. After each round of the experiment, excrement such as feces and urine were removed in time, the experimental device was wiped with 75% alcohol, and the experimental device was dried to remove the odor left by the previous animal as much as possible, and then the next mouse was replaced for behavioral testing.

[0156] The social novelty preference index (SPI) was calculated as follows: SPI = (time spent in stranger mouse 2 area) / (time spent in stranger mouse 1 area + time spent in stranger mouse 2 area).

[0157] 8.5 Self-grooming test - used to detect repetitive stereotyped behaviors in male mice

[0158] One of the core behavioral disorders of ASD is repetitive stereotyped behavior, which is specifically manifested in human children as some high-frequency and meaningless behaviors, difficulty accepting changes in things, and narrow interests. In the VPA-induced ASD rat model, it is often manifested as meaningless chewing, repeated digging, repeated hair combing, vertical jumping and other behaviors. On the 32nd day after birth, the test mice were first placed in the open field box to freely explore and adapt to the environment for 10 minutes, and then the timing was started for 10 minutes to observe the cumulative time that the test mice used their claws or mouths to comb their faces, limbs, bodies and tails.

[0159] 8.6 Marble Burial Test (Bead Burial Test)

[0160] The buried bead test is a classic experiment that can quantitatively detect repetitive stereotyped behaviors in VPA-induced ASD rat models. The test mice were placed in a box. A layer of clean bedding was laid on the bottom of the box, about 5 cm thick. Before the test mice officially started the experiment, they were placed in a box with bedding to adapt for 3 minutes in advance, and then the mice were taken out and placed in a transfer cage to wait. The bedding in the box was laid flat, and 16 black glass balls with a diameter of 1.6 mm were placed in a 4×4 grid. Then the test mice were placed in and allowed to move freely for 10 minutes. After 10 minutes, the test mice were taken out and photographed from various angles with a camera. Finally, three trained statisticians who were not related to this subject calculated the number of buried beads from the pictures and videos: more than 75% of the glass beads were buried in the bedding and were considered buried. The statisticians were unaware of the grouping situation, and the final results were averaged and rounded.

[0161] 8.7 Rat Tail Light Pain Measurement Experiment

[0162] The rat tail light pain measurement experiment uses heat to stimulate the rat's tail. When the tail is subjected to harmful stimulation, it will produce an obvious avoidance reaction. It can test lightly anesthetized animals and is not affected by the animal's movement coordination. Therefore, it has certain advantages over the hot plate test.

[0163] ① Take the experimental animals out of the animal room, weigh them, let them adapt to the laboratory for 30 minutes, and separate the control group from the model group;

[0164] ② Determine the baseline latencies of animals using a tail flash test instrument (i.e., there is a heating light source under a small hole in the plate, place the tail of a rat (about 50 mm in front of the tail tip) or a mouse (about 15 mm in front of the tail tip) above the small hole, start the heating light source and start timing until the tail dodges. Adjust the intensity of the light source and set the tail dodge time of most mice to 3-4 seconds. If there is no dodge reflex, set the test end time to 10 seconds to avoid burns;

[0165] ③ Test the tail flash reaction. Place the animal on the tail flash test board and place its tail on the light source hole. Start timing and observe the time for the animal's tail to avoid the reaction, or until the cut-off time. Note that only one time point is measured in one test, not three time points.

[0166] Notes: a. If the animal does not react or the reaction latency is very close to the end time of the test, it means that the stimulation intensity is too weak (the light source temperature is set low), and the stimulation intensity must be increased. b. All experimental procedures must be kept consistent, and the animals must be caught as lightly as possible to allow the animals enough time to adapt to the experimenter's smell and laboratory environment, otherwise it may cause significant fluctuations in the experimental results or be impossible to repeat. c. The intensity of the stimulus light source, that is, the intensity of the stimulus is too high or too weak, can cause large changes in the test results, and high-intensity stimulation may mask the reaction differences between animals, so the stimulation program must be set reasonably. d. In order to reduce experimental errors, the activities of animals should generally be restricted appropriately.

[0167] The observation results are as follows:

[0168] 1 Response, mortality and abortion rate of C57BL / 6 pregnant mice in each group after administration of VPA at different doses and time

[0169] After a single intraperitoneal injection of VPA 600 mg kg-1, the C57BL / 6 pregnant mice in the traditional model group developed limb stiffness, difficulty walking, closed eyes and paralysis in the mouse cage, or even heartbeat and breathing disorder, which eventually led to death within 3-5 minutes; while after three administrations at different times in the modified group 1 and the modified group 2, it was obviously lighter than the traditional group. The limb stiffness of the mice in the modified group 1 appeared about 13-15 minutes after administration, and they were still able to walk within the 30 minutes of the entire observation period. The limb stiffness of the C57BL / 6 mice in the modified group 2 appeared about 8-10 minutes after administration, and they were still able to walk within the 30 minutes of the entire observation period. Within 6 hours after administration, the mortality rates of the modified group 1 and the modified group 2 were both 0%, and the mortality rate of the traditional group was 33.3%. The mortality rates of the modified group 1 and the modified group 2 were significantly lower than those of the traditional group (0% vs 33.3%, see Table 1), and there were significant differences between the groups (P<0.01). Abortion rate: The abortion rate of improved group 1 was significantly lower than that of the traditional group (33.3% vs 50%, see Table 1), and there was a significant difference between the groups (P<0.01). The abortion rate of improved group 2 was significantly lower than that of the traditional group (16.7% vs 50%, see Table 1), and there was a significant difference between the groups (P<0.01).

[0170] Table 1 Comparison of the production of pregnant mice

[0171]

[0172] 2 The rate of appearance deformity in offspring of C57BL / 6 pregnant mice in each group after administration of VPA at different doses and time

[0173] All newborns in the normal control group of C57BL / 6 mice had ruddy skin, intact body shape, and no developmental deformities. Some of the newborns in the traditional model group, which was given VPA 600 mg kg-1 once by intraperitoneal injection, had short limbs, unclear eyeballs and auricles, and even had only one eyeball. The appearance deformity rate of some of the newborns in the traditional model group was 31.3% (5 / 16), which was significantly higher than that in the normal control group (0% (0 / 45)) (P<0.01), indicating that high-dose VPA exposure by intraperitoneal injection during pregnancy has serious embryotoxicity to pregnant C57BL / 6 mice; while the newborns of C57BL / 6 mice in the modified group 1 and modified group 2, which were given VPA by intraperitoneal injection in batches according to different doses and time, had no obvious appearance deformities.

[0174] 3 Physiological developmental indicators of C57BL / 6 male offspring in each group after administration of VPA at different doses and times

[0175] The 4-day survival rate of C57BL / 6 male pups born in the traditional model group was 80% (8 / 10) after a single intraperitoneal injection of VPA 600 mg kg-1, and the 4-day survival rate of C57BL / 6 male pups born in the control group and the modified group 1 and modified group 2 was 100% (Table 2). The body weight and tail length of C57BL / 6 pups in each group were measured on days 7, 14, 21, and 28 after birth. The body weight of C57BL / 6 male pups born in the modified group 1 and modified group 2 was lighter than that of the control group from day 7 to 28 after birth (P<0.001); the body weight of male pups born in the traditional group was lower than that of the control group from day 14 after birth (P<0.01). Brachytail deformity in rodents is believed to be associated with mild neural tube defects. The tail length of male offspring born in the modified group 1, modified group 2 and traditional group C57BL / 6 mice was shorter than that in the control group (P<0.05). The incidence of bent tails in male offspring born in the traditional group C57BL / 6 mice was 37.5%, and most of the male offspring born in the modified group 1 and modified group 2 had tail bending to varying degrees, with the incidence of bent tails in the modified group 1 and modified group 2 being 81.3% and 90% respectively. The bent tail rate of male offspring born in the modified group 1 and modified group 2 was significantly higher than that in the traditional model group given VPA 600 mg kg-1 once by intraperitoneal injection (P<0.01). (Table 2).

[0176] Table 2 Mortality rate of newborn mice and tail bending rate of offspring mice

[0177]

[0178] 4 The age at which the incisor eruption, eye opening and abdominal hair growth of C57BL / 6 male offspring in each group reached positive after VPA administration at different doses and times

[0179] (1) Figure 1 As shown in A and B, the days of positive incisor eruption and eye opening of male offspring born to C57BL / 6 pregnant mice in modified group 1 and modified group 2 were significantly delayed compared with those in the normal control group (P<0.01). The days of positive incisor eruption of male offspring born to C57BL / 6 pregnant mice in the traditional model group were not significantly different from those in the normal control group (P>0.05). (2) Figure 1 As shown in C, the days when the abdominal hair growth of male offspring born to C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 reached positive growth were not significantly different from those in the normal control group (P>0.05). This suggests that VPA injection during pregnancy can significantly delay the physiological development of male offspring born to C57BL / 6 pregnant mice, such as incisor eruption and eye opening.

[0180] 5 Development of the nervous system of C57BL / 6 male offspring after administration of VPA at different doses and times

[0181] The results of nerve reflex function test showed that ( Figure 2 ): (1) Compared with the normal control group, the turning behavior of male offspring born to C57BL / 6 pregnant mice in the traditional model group and improved group 1 and improved group 2 reached positive time later (P<0.05); (2) Compared with the normal control group, the plane righting reflex of male offspring born to C57BL / 6 pregnant mice in the traditional model group and improved group 1 and improved group 2 was Figure 2 Middle A), aerial righting reflex ( Figure 2 Middle B) and cliff avoidance reflex ( Figure 2 Middle C) There was no significant difference in the time it took for the behavior to reach positive (P>0.05).

[0182] 6 Observation of motor coordination ability and muscle tension of male offspring born from C57BL / 6 pregnant mice in each group after administration of VPA at different doses and times

[0183] Compared with the normal control group, the straight-line crawling behavior of male offspring born from C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 was Figure 3 There was no significant difference in the time to reach positivity in A, but the grasping behavior ( Figure 3 (B) The time to reach positivity was significantly delayed (P<0.05), indicating that VPA exposure in mid-gestation had no significant effect on the motor coordination function of male offspring born from C57BL / 6 pregnant mice, but could lead to decreased muscle tone.

[0184] 7 Observation of auditory startle reflex in male offspring of C57BL / 6 pregnant mice after administration of VPA at different doses and times

[0185] like Figure 4 As shown in the data, compared with the normal control group, the time for the auditory startle reflex to become positive in male pups born from C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 was significantly earlier than that in the normal control group (P<0.01), indicating that VPA exposure in mid-gestation may lead to auditory hypersensitivity in male pups born from C57BL / 6 pregnant mice.

[0186] 8 Effects of different doses and time of VPA administration on spatial exploration and anxiety of male offspring born from C57BL / 6 pregnant mice

[0187] The open field test is mainly used to detect the autonomous behavior, exploratory activities and anxiety level of experimental animals in unfamiliar environments. (1) In the open field test, compared with the normal control group, the male offspring of C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 showed significant differences in the number of central grids crossed (P<0.05), indicating that the male offspring of C57BL / 6 pregnant mice exposed to VPA showed obvious anxiety behavior ( Figure 5(2) Compared with the normal control group, the vertical scores of male offspring born from C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 were lower in the open field than those in the normal control group (P<0.05) ( Figure 5 (3) There was no significant difference in the number of defecation of male pups born to C57BL / 6 pregnant mice in the traditional model group, modified group 1, and modified group 2 compared with the normal control group, indicating that VPA exposure during mid-gestation does not lead to increased anxiety in male pups born to C57BL / 6 pregnant mice. Figure 5 C and D show representative trajectory graphs of male pups in the control group in the open field test and representative trajectory graphs of male pups in the improved group 2 in the open field test.

[0188] 9Social ability and interest preference of male offspring born from C57BL / 6 pregnant mice in each group after administration of VPA at different doses and times

[0189] The three-box social experiment mainly tests the social ability of the test animals and their preference for novel things. The first stage of the three-box social experiment is mainly used to test social tendency, which is defined as the test mice preferring to move around the cage with strange mice rather than around the same empty cage. The detection goal of this stage is mainly to quantify the test mice's interest in social cues. In the first stage of the three-box social experiment, compared with the normal control group, the time that the male offspring born to C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 stayed in the box with Rat 1 (Stranger 1cage) and the time of mutual sniffing contact with Rat 1 (Stranger 1) were significantly reduced (P<0.01); compared with the normal control group, the time that the male offspring born to C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 sniffed the object (Object) was also significantly reduced (P<0.05); compared with the normal control group, there was no significant difference in the time that the male offspring born to C57BL / 6 pregnant mice in the traditional model group, modified group 1 and modified group 2 stayed in the middle compartment (P>0.05), but the time spent exploring and staying in the object cage (Object cage) was significantly increased (P<0.01), and the male offspring born to C57BL / 6 pregnant mice showed unwillingness to interact with the stranger mouse (Stranger 1) to communicate, and preferred to stay in a space without strange mice, suggesting that VPA exposure in mid-pregnancy will lead to a significant decrease in the social ability of male offspring born from C57BL / 6 pregnant mice (P<0.01) ( Figure 6 (A).

[0190] In the three-box social experiment, the second phase of the experiment was mainly used to test the preference for social novelty. Figure 6B), which is defined as a tendency to move more around the cage with Stranger 2 rather than interacting with the same mice encountered in the first stage of the experiment. The detection goal of this stage is mainly to quantify the interest of the tested mice in new social cues. In the second stage of the experiment, Stranger 2 was placed in the object box to detect the preference of each group of animals for novel things. Compared with the control group, the time spent exploring and staying in the box of Stranger 1 (P<0.01) and the time spent on mutual olfactory contact communication with Stranger 1 (P<0.01) of male pups born from C57BL / 6 pregnant mice in the traditional model group and the modified group 1 and modified group 2 was significantly increased, and the time spent exploring and staying in the box of Stranger 2 (P<0.05) was significantly reduced, but there was no significant difference in the time spent on mutual olfactory contact communication with Stranger 2 (P>0.05). This indicates that male offspring born from C57BL / 6 pregnant mice have narrow interests and do not have a clear tendency to "get bored with the old and love the new".

[0191] Figure 6 C and D in the figure show the representative trajectory graphs of male pups in the control group and the representative trajectory graphs of male pups in the improved group 2 in the three-box social experiment of mice.

[0192] 10 Others

[0193] In the self-stroking experiment, the results showed that the male offspring of C57BL / 6 mice in the model group showed a significantly longer self-stroking time than the normal control group ( Figure 7 A), the number of buried marbles increased significantly ( Figure 7 Middle B), showing obvious repetitive stereotyped behaviors, similar to those of patients with autism spectrum disorder.

[0194] Example 2 Preparation of VPA-induced ASD animal model - SD rats

[0195] SD rats were randomly divided into 4 groups according to the VPA administration method:

[0196] (1) Traditional model group:

[0197] SD rats were given VPA 600 mg kg intraperitoneally at 12.5 days of gestation. -1 .

[0198] (2) Improved model group 1:

[0199] SD rats were given VPA 300 mg kg by intraperitoneal injection at 11 days of gestation. -1 ;

[0200] At 12.5 days of gestation, VPA 450 mg kg was given intraperitoneally once. -1 ;

[0201] At 13 days of gestation, VPA 300 mg kg was given once by intraperitoneal injection. -1 .

[0202] (3) Improved model group 2:

[0203] SD rats were given VPA 400 mg kg intraperitoneally at 11.5 days of gestation. -1 ;

[0204] At 12.5 days of gestation, VPA 450 mg kg was given intraperitoneally once. -1 ;

[0205] At 13.5 days of gestation, VPA 400 mg kg was given by intraperitoneal injection once -1 .

[0206] (4) Normal saline control group: SD rats were given a single injection of 600 mg / kg normal saline at 12.5 days of gestation. -1 .

[0207] After group drug administration:

[0208] ①Observe the adverse reactions, mortality and abortion rate of pregnant SD rats in each group after administration of sodium valproate or normal saline;

[0209] ②Observe the morphology, growth and development of the newborn mice in each group;

[0210] ③ Observe the social behavior of each group of newborn mice, for example: including three-box social behavior experiments, youth play experiments and open field experiments, etc., various indicators reflecting autism-like behaviors of the mice.

[0211] The specific observation method is the same as in Example 1, and the observation results are as follows:

[0212] 1. Response, mortality and abortion rate of pregnant SD mice in each group after administration of VPA at different doses and time

[0213] The SD pregnant mice in the traditional group developed limb stiffness, difficulty walking, and paralysis in the mouse cage with eyes closed within 7 minutes after administration; while the reactions of the modified group 1 and modified group 2 were significantly lighter than those of the traditional group. The limb stiffness of the SD mice in the modified group 1 appeared about 15-18 minutes after administration, and they were able to walk within the 30 minutes of the entire observation period. The limb stiffness of the SD mice in the modified group 2 appeared about 12-15 minutes after administration, and they were able to walk within the 30 minutes of the entire observation period. Within 6 hours after administration, the mortality rate of the modified group 1 and modified group 2 was 0%, and the mortality rate of the traditional group was 16.7%. The mortality rate of the modified group 1 and modified group 2 was significantly lower than that of the traditional group (0% vs 16.7%, see Table 3), and there was a significant difference between the groups (P<0.05). Abortion rate: The abortion rate of the modified group 1 was significantly lower than that of the traditional group (16.7% vs 50%, see Table 3), and there was a significant difference between the groups (P<0.05). The abortion rate in the modified group 2 was significantly lower than that in the traditional group (0% vs 50%, see Table 3), and there was a significant difference between the groups (P<0.01).

[0214] Table 3 Comparison of the production of pregnant mice

[0215]

[0216] 2 The rate of appearance deformity of offspring born by SD pregnant mice in each group after VPA administration at different doses and time

[0217] All newborns in the normal control group of SD rats had ruddy skin and intact body shape without any limb malformation. Some of the newborns in the traditional model group, which was given VPA 600 mg kg-1 once by intraperitoneal injection, showed deformities such as short limbs, unclear eyeballs and auricles. The appearance deformity rate of some newborns in the traditional model group was 6.5% (2 / 31), which was not significantly different from the normal control group (0% (0 / 78)) (P>0.05); while the newborns of SD rats in the modified group 1 and modified group 2, which were given VPA by intraperitoneal injection in batches according to different doses and time, showed no obvious appearance deformity, and the appearance deformity rate of the newborns was 0%.

[0218] 3 Physiological developmental indicators of male SD rats in each group after VPA administration at different doses and times

[0219] The 4-day survival rate of male SD mice born in the traditional model group was 64.3% (9 / 14) after a single intraperitoneal injection of VPA 600 mg kg-1. The 4-day survival rate of male SD mice born in the control group and the modified groups 1 and 2 was 100% (Table 4), which was significantly different from the 4-day survival rate of male SD mice born in the traditional model group (P<0.01). The body weight and tail length of SD mice in each group were measured on days 7, 14, 21, and 28 after birth. The body weight of male SD mice born in the modified groups 1 and 2 was lighter than that of the control group from day 7 to 28 after birth (P<0.001); the body weight of male mice born in the traditional group was lower than that of the control group from day 14 after birth (P<0.01). Short and deformed tails in rodents are considered to be related to mild neural tube defects.

[0220] The tail length of male offspring born in the modified group 1, modified group 2 and traditional group was shorter than that in the control group (P<0.05). The incidence of bent tails in male offspring born in the traditional group was 55.6%, and most of the male offspring born in the modified group 1 and modified group 2 had tail bending to varying degrees, with the incidence of bent tails in the modified group 1 and the modified group 2 being 90.3% and 95.3% respectively. The bent tail rate of male offspring born in the modified group 1 and modified group 2 was significantly higher than that in the traditional model group given VPA 600 mg kg-1 once by intraperitoneal injection (P<0.01) (Table 4).

[0221] Table 4 Mortality rate of newborn mice and tail bending rate of offspring mice

[0222]

[0223] 4 The age at which the incisor eruption, eye opening and abdominal hair growth of male SD rats in each group reached positive after VPA administration at different doses and times

[0224] The days of positive incisor eruption and eye opening of male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 were significantly delayed compared with those in the normal control group (P<0.01) ( Figure 8 A and B); The days when the abdominal hair growth of male offspring born from C57BL / 6 pregnant mice in the traditional model group, improved group 1 and improved group 2 reached positive were significantly delayed compared with those in the normal control group (P<0.01) ( Figure 8 C). This suggests that VPA injection during pregnancy can significantly retard the physiological development of male offspring born to SD pregnant mice, such as incisor eruption, eye opening, and abdominal hair growth.

[0225] 5. Development of the nervous system of male offspring born from pregnant SD mice in each group after administration of VPA at different doses and times

[0226] The results of nerve reflex function test showed that ( Fig. 9): (1) Compared with the normal control group, the time for the turning behavior of male offspring born to SD pregnant mice in the traditional model group and improved group 1 and improved group 2 to reach positive was significantly delayed compared with the normal control group (P<0.01); (2) Compared with the normal control group, the plane righting reflex ( Fig. 9 Middle A), aerial righting reflex ( Fig. 9 Middle B) and cliff avoidance reflex ( Fig. 9 Middle C) The time for the behavior to become positive was significantly delayed compared with the normal control group (P<0.01). This suggests that the developmental delay of the neuroreflex function of male offspring born from SD pregnant mice injected with VPA in the second trimester is more obvious than that of male offspring born from C57BL / 6 pregnant mice.

[0227] 6 Observation of motor coordination ability and muscle tension of male offspring born from SD pregnant mice in each group after administration of VPA at different doses and times

[0228] Compared with the normal control group, the time for the straight-line crawling behavior of male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 to reach positive ( Fig.10 A) and the time to positive grasping behavior ( Fig.10 The results in Figure 3 (B) were significantly delayed compared with the normal control group (P<0.01). The results suggest that VPA exposure in mid-gestation not only impaired the motor coordination ability of male offspring born from SD pregnant mice, but also affected the muscle tone.

[0229] 7 Observation of auditory startle reflex of male offspring born from SD pregnant mice in each group after administration of VPA at different doses and times

[0230] like Fig.11 As shown in the data, compared with the normal control group, the time for the auditory startle reflex of male pups born to SD pregnant mice in the traditional model group, improved group 1 and improved group 2 to become positive was significantly earlier than that in the normal control group (P<0.01), suggesting that VPA exposure in mid-gestation may lead to auditory hypersensitivity in male pups born to SD pregnant mice.

[0231] 8 Effects of VPA administration at different doses and times on spatial exploration and anxiety of male offspring born from SD pregnant mice in each group

[0232] The open field test is mainly used to detect the autonomous behavior, exploratory activities and anxiety level of experimental animals in unfamiliar environments. (1) In the open field test, compared with the normal control group, there was no significant difference in the total number of male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 ( Fig.12(A), indicating that VPA exposure does not affect the motor ability of male offspring born from SD pregnant mice; (2) Compared with the normal control group, the central grid crossing number and vertical score of male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 were lower than those in the normal control group (P<0.01)( Fig.12 (B) This indicates that VPA exposure during mid-pregnancy can lead to a decrease in the exploration interest of male pups born to SD pregnant mice in unfamiliar environments; (3) There was no significant difference in the number of defecations of male pups born to SD pregnant mice in the traditional model group, modified group 1, and modified group 2 compared with the normal control group, indicating that VPA exposure during pregnancy will not lead to increased anxiety in male pups born to SD pregnant mice.

[0233] Fig.12 C and D in the figure show the representative trajectory graphs of male pups in the control group and the representative trajectory graphs of male pups in the improved group 2 in the open field test.

[0234] 9Social ability and interest preference of male offspring born from SD pregnant mice in each group after administration of VPA at different doses and times

[0235] The three-box social experiment mainly tests the social ability of the test animals and their preference for novel things. In the first stage of the three-box social experiment, compared with the normal control group, the time that the male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 stayed in the box with Rat 1 (Stranger 1cage) and the time of mutual sniffing contact with Rat 1 (Stranger 1) were significantly reduced (P<0.01); compared with the normal control group, the time that the male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 sniffed with the object (Object) was also significantly reduced (P<0.05); compared with the normal control group, there was no significant difference in the time that the male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 stayed in the middle compartment (P>0.05), but the time spent exploring and staying in the object cage (Object cage) was significantly increased (P<0.01), and the male offspring born from SD pregnant mice showed unwillingness to interact with the stranger mouse (Stranger 1). 1) to communicate, but preferred to stay in a space without strange mice, suggesting that VPA exposure during mid-pregnancy will lead to a significant decrease in the social ability of male offspring born from SD pregnant mice (P<0.01) ( Fig.13 In the second stage of the experiment, Stranger 2 was placed in the object box to test the preference of each group of animals for novel things ( Fig.13Middle B). Compared with the control group, the male offspring born from SD pregnant mice in the traditional model group, modified group 1 and modified group 2 spent significantly more time exploring and staying in the familiar mouse 1 (Stranger 1 cage) (P < 0.01) and the time spent on sniffing and communicating with the familiar mouse 1 (Stranger 1) (P < 0.01), and the time spent exploring and staying in the strange mouse 2 (Stranger 2 cage) and the time spent on sniffing and communicating with the strange mouse 2 (Stranger 2) (P < 0.01) were significantly reduced. This shows that male offspring born from SD pregnant mice have the characteristics of narrow interests and no obvious "love the new and hate the old", and the characteristics of narrow interests are more obvious than those of male offspring born from C57BL / 6 pregnant mice.

[0236] Fig.13 C and D in the figure show the representative trajectory graphs of male pups in the control group and the representative trajectory graphs of male pups in the improved group 2 in the three-box social experiment of rats.

[0237] 10 Others

[0238] In the self-stroking experiment, the results showed that the male offspring born to SD rats in the model group showed a significantly longer self-stroking time than that in the normal control group ( Fig.14 A), the number of buried marbles increased significantly ( Fig.14 Middle B), showing obvious repetitive stereotyped behaviors, similar to those of patients with autism spectrum disorder.

[0239] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing an autism animal model, comprising administering sodium valproate to the test animal three times when the test animal is 11-14 days pregnant, wherein the dose of sodium valproate administered to the test animal each time is less than 600 mg·kg -1 , the test animals are rodents in a pregnant state.

2. The establishment method according to claim 1, characterized in that: When the test animals were 12.5 days pregnant, the test animals were administered sodium valproate for the second time, the first administration of sodium valproate to the test animals was 0.5 days or more from the second administration of sodium valproate to the test animals, and the third administration of sodium valproate to the test animals was 0.5 days or more from the second administration of sodium valproate to the test animals.

3. The establishment method according to claim 1, characterized in that: The test animals are rats and / or mice.

4. The establishment method according to claim 1, characterized in that: When the test animal is a rat, the dose of sodium valproate administered to the test animal each time is 300-500 mg·kg -1 ; Preferably, when the test animal is a mouse, the dose of sodium valproate administered to the test animal each time is 200-400 mg·kg -1 .

5. The establishment method according to claim 4, characterized in that: The method comprises administering sodium valproate to rats at 11-14 days of gestation in three doses, wherein: At 11 days of pregnancy, 300 mg / kg -1 Sodium valproate was given at a dose of ; At 12.5 days of pregnancy, 450 mg / kg -1 Sodium valproate was given at a dose of ; At 13 days of pregnancy, 300 mg / kg -1 Sodium valproate was given at a dose of 6. The establishment method according to claim 4, characterized in that: The method comprises administering sodium valproate to rats at 11-14 days of gestation in three doses, wherein: At 11.5 days of gestation, 400 mg / kg -1 Sodium valproate was given at a dose of ; At 12.5 days of pregnancy, 450 mg / kg -1 Sodium valproate was given at a dose of ; At 13.5 days of pregnancy, 400 mg / kg -1 Sodium valproate was given at a dose of 7. The establishment method according to claim 4, characterized in that: The method includes administering sodium valproate to mice three times when the mice are 11-14 days pregnant, At 12 days of pregnancy, 200 mg / kg -1 Sodium valproate was given at a dose of ; At 12.5 days of pregnancy, 300 mg / kg -1 Sodium valproate was given at a dose of ; At 13 days of pregnancy, 200 mg / kg -1 Sodium valproate was given at a dose of 8. The establishment method according to claim 4, characterized in that: The method includes administering sodium valproate to mice three times when the mice are 11-14 days pregnant, At 11.5 days of gestation, 300 mg / kg -1 Sodium valproate was given at a dose of ; At 12.5 days of pregnancy, 400 mg / kg -1 Sodium valproate was given at a dose of ; At 13.5 days of pregnancy, 300 mg / kg -1 Sodium valproate was given at a dose of 9. An autism animal model obtained by the method according to any one of claims 1 to 8.

10. Use of the establishment method according to any one of claims 1 to 8 or the autism animal model according to claim 9 in any one of the following: Study the causes and / or pathogenesis of autism; Prepare products for studying the causes and / or pathogenesis of autism; Screening for drugs to prevent and / or treat autism; Preparation of products for screening drugs for the prevention and / or treatment of autism; Prepare products for studying the mechanism of action and / or therapeutic efficacy of autism treatments.

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