Method for inducing rat autism model by using neurotoxin

Abstract

A method for inducing a rat autism model by using a neurotoxin, relating to the technical field of animal model construction. Provided is a method for inducing a juvenile rat autism model by using a neurotoxin. A female rat is injected with β-N-methylamino-L-alanine (BMAA) and then caged with a male rat, and the offspring produced is a juvenile rat autism model; a normally born juvenile rat is injected with BMAA, and a juvenile rat autism model is obtained. The use of a neurotoxin BMAA to establish an autism model can reproduce a mother-to-child transmission process of BMAA, and can also simulate the process by which a newborn is exposed postnatally to the toxin BMAA in an environment, leading to disease onset. The method for constructing an animal model has a relatively simple preparation process, causes few abnormalities, and is beneficial to subsequent research on diagnosis and treatment of autism.

Description

A method for inducing a rat autism model by using a neurotoxin TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal model construction, and particularly relates to a method for inducing a rat autism model by using a neurotoxin. BACKGROUND

[0002] Autism, also known as autism, is a developmental neuropsychiatric disease that often occurs in adolescents. Patients often show abnormal behaviors and emotions such as social impairment, repetitive stereotyped movements, and anxiety. It is also a pervasive developmental disorder, and the main causes include genetic and environmental factors. Studies have shown that autism is highly related to genetic factors, in addition, including brain structure and function abnormalities, biological mechanisms, and adverse factors during the mother's pregnancy, etc. can also affect the onset of autism. Exposure to chemicals during the prenatal and neonatal periods can cause central nervous system development abnormalities.

[0003] In recent years, the prevalence of autism in countries around the world has increased year by year, and the basic and clinical research of autism and the research of autism animal models have become one of the hotspots in the field of pre-medical and neuroscience. At present, in the international range, autism has not only become a medical problem, but also gradually become a social problem that needs to be solved under the medical background. Therefore, it is particularly important to improve the cognitive and research level of the disease. Animal models have the advantages of controllability, availability, and predictability, which provide advantages over human research, and play an important role in the exploration of disease causes and pathogenesis. Autism animal models have become the key to exploring the relationship between the nervous system and the onset of autism.

[0004] At present, the animal models created around the causes and pathogenesis of autism are increasing, which on the one hand shows the academia's continuous attention to the disease, and on the other hand the diversification of the models also causes no consensus in research. Looking at the evaluation standards of animal models at home and abroad, three kinds of effectiveness are widely used to evaluate the closeness of the model to human disease. The more effective the model presents, the better it presents the effect of human disease. These effectiveness is not only crucial in assessing the reliability of animal models, but also irreplaceable in evaluating the efficacy of drug treatment. In 2008, at the neurotoxicology conference, clinicians and basic research scientists from various countries attended a special symposium on autism to discuss the behavioral characteristics of autism and how to use neurotoxicology to establish an autism model in rodents or primates. The current common autism model preparation methods include neurotoxicology model and genetic model.

[0005] Autism spectrum disorder is currently considered to be caused by the combined effects of environmental and genetic factors. Epidemiological studies have shown that exposure of the fetus to certain neurotoxic drugs, such as antiepileptic drugs such as valproic acid (VPA) and propionic acid, can cause developmental disorders of the nervous system, severely affect the development of cognitive function, and lead to autism. Therefore, neurotoxicological methods are one of the important means for establishing and studying animal models of autism. Prenatal VPA exposure is a classic and commonly used neurotoxicological autism model. After intraperitoneal injection of VPA to pregnant mice at 12.5 days of pregnancy, the offspring of the mice can exhibit autism spectrum disorder-like behaviors such as reduced social and exploratory behavior, repetitive stereotyped movement, and anxiety and tension.

[0006] SUMMARY

[0007] The purpose of the present application is to provide a method for inducing a rat autism model using a neurotoxin. The established autism model can reproduce the maternal-fetal transmission process of BMAA and can also simulate the process of a newborn contacting BMAA toxin in the environment after birth, leading to the occurrence of the disease. The preparation process is relatively simple and can be completed without the need for many reagents and equipment support. The operation process is simple and easy to operate, and abnormal situations are less likely to occur.

[0008] The present application provides the use of beta-N-methylamino-L-alanine in the preparation of an autism animal model.

[0009] The present application also provides a method for inducing a rat pup autism model using a neurotoxin, which comprises injecting beta-N-methylamino-L-alanine into a female rat and mating the female rat with a male rat. The offspring of the female rat is a rat pup autism model.

[0010] Or injecting beta-N-methylamino-L-alanine into a normal postnatal rat pup to obtain a rat pup autism model.

[0011] Preferably, the injection comprises injecting a PBS solution of beta-N-methylamino-L-alanine.

[0012] Preferably, the injection comprises injecting beta-N-methylamino-L-alanine into a female rat at a dose of 1-1000 mg / kg body weight.

[0013] The injection comprises injecting beta-N-methylamino-L-alanine into a pup at a dose of 1-200 mg / kg body weight.

[0014] Preferably, the method of injection comprises subcutaneous injection.

[0015] Preferably, the subcutaneous injection is performed once.

[0016] Preferably, after obtaining the rat pup autism model, the method further comprises verifying the rat pup autism model using an open field test.

[0017] Preferably, the rat infant autism model obtained by the method of injecting the female mouse is subjected to the open field test after the infant is born for 3-6 weeks to determine whether the infant has autism behavior.

[0018] Preferably, the rat infant autism model obtained by the method of injecting the infant is subjected to the open field test after 1-2 weeks of injection to determine whether the infant has autism behavior.

[0019] Preferably, the rat includes a Wistar rat, an SD rat, an F344 rat, a Lewis rat, an SHR rat or a WKY rat.

[0020] Beneficial effects: the present application provides a method for inducing a rat infant autism model by using a neurotoxin. The rat infant autism model can be prepared by two methods: a female mouse is injected with β-N-methylamino-L-alanine (BMAA), and then is caged with a male mouse, and the offspring of the female mouse is the rat infant autism model; or a normally born infant is injected with BMAA to obtain the rat infant autism model.

[0021] The present application uses the neurotoxin BMAA to establish an autism model, which is more conducive to subsequent research on autism diagnosis and treatment, and has advantages that previous animal models do not have. In the embodiments of the present application, the female mouse is caged after being administered with different doses of BMAA, and the toxin content in the blood of the mother mouse before and after administration and the infant born by the mother mouse administered with BMAA is detected to obtain the metabolic accumulation of BMAA in the mother mouse and the accumulation of toxins in the infant. The autism model established in the present application can reproduce the mother-infant transmission process of BMAA, and can also simulate the process of a newborn contacting BMAA toxin in the environment after birth, leading to the occurrence of the disease. The construction method of the animal model described in the present application has a relatively simple preparation process, which can be completed without the support of many reagents and equipment instruments, and the operation process is simple and easy to operate, and abnormal situations are less likely to occur. BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a process diagram of rat feeding;

[0023] FIG. 2 is a process diagram of female rat administration;

[0024] FIG. 3 is a process diagram of infant open field test;

[0025] FIG. 4 is a motion trajectory heat map of the infant born by the high-dose mother mouse group in Example 1, wherein the right side numbers in the figure represent the time (unit: s) that the infant stays at a certain position, and the same applies below;

[0026] FIG. 5 is a motion trajectory heat map of the infant born by the low-dose mother mouse group in Example 1;

[0027] Figure 6 is a motion trajectory heat map of the pups produced by the control group of female rats in Example 1;

[0028] Figure 7 is a motion trajectory heat map of the control group of pups in Example 2;

[0029] Figure 8 is a motion trajectory heat map of the test group of pups in Example 2. DETAILED DESCRIPTION

[0030] The present application provides an application of β-N-methylamino-L-alanine in preparing an autism animal model.

[0031] The β-N-methylamino-L-alanine described in the present application is referred to as BMAA.

[0032] The present application also provides a method for inducing a rat pup autism model using a neurotoxin, comprising injecting a female rat with β-N-methylamino-L-alanine and then co-caging the female rat with a male rat, and the pups born are the rat pup autism model;

[0033] or injecting a normal postnatal rat pup with β-N-methylamino-L-alanine to obtain the rat pup autism model.

[0034] The rat pup autism model described in the present application can be constructed by two routes, (1) by co-caging the female rat after prenatal administration of BMAA to produce pups, obtaining the rat pup autism model, (2) by injecting BMAA to the postnatal pups, obtaining the rat pup autism model.

[0035] The rats described in the present application preferably include Wistar rats, SD rats, F344 rats, Lewis rats, SHR rats, or WKY rats. The injection described in the present application preferably includes injection of a PBS solution of BMAA, and the concentration of the PBS solution of BMAA for injection is calculated according to the required dose of injection volume and rat body weight, and the PBS solution of BMAA for injection is prepared in a concentration range of 0.1-100 mg / ml, preferably 0.1-15 mg / ml. The volume of the PBS solution of BMAA for injection preferably ranges from 0.01 to 5 ml, more preferably from 0.1 to 2 ml; the injection amount is preferably 1-1000 mg / kg of body weight of rats, more preferably 1-50 mg / kg of body weight of rats. The PBS solution of BMAA described in the present application preferably includes a PBS solution of BMAA with different concentrations prepared by adding a purchased BMAA freeze-dried powder to a PBS solution. The BMAA described in the examples of the present application is preferably purchased from Beijing Inokai Technology Co., Ltd., batch number: 150053, product name: β-N-methylamino-L-alanine hydrochloride, which is a BMAA hydrochloride salt. The PBS solution described in the present application is preferably purchased from Biological Industries (BI), batch number: 2246306, concentration: 0.01 M.

[0036] The PBS solution of BMAA is injected into female rats in the present application, and the injection dose of adult rats does not exceed 1.64 g / kg of body weight, and the amount of BMAA injected into adult female rats is preferably 1-1000 mg / kg of body weight, more preferably 1-15 mg / kg of body weight of rats.

[0037] When the model is prepared directly using postnatal pups in the present application, drug injection can be performed immediately after birth, but the body condition of the pups needs to be observed, and the time for behavioral testing is also required, so the administration time is from birth to 1-2 weeks before the behavioral testing experiment, preferably the administration time is 3-4 weeks after birth, more preferably the time is 25 days after birth. When injecting into pups, the PBS solution of BMAA is also injected, and the injection amount is calculated based on BMAA, and the injection dose of pups does not exceed 3.65 g / kg of body weight, and the amount of BMAA injected into pups is preferably 1-200 mg / kg of body weight, more preferably 50 mg / kg of body weight of rats. When the subcutaneous injection described in the present application is performed, it is preferably injected once.

[0038] After the rat infant autism model is constructed by the two methods, the open field experiment is preferably used for verification. Specifically, the model is constructed by the method of injecting the female mice, and the open field experiment can be performed on the infant mice after 3-6 weeks of birth, and the optimal time is 4-5 weeks. The open field experiment can reflect the autism behavior of the infant mice. The autism model of the infant mice is obtained by the method of injecting the infant mice, and the open field experiment is performed after 1-2 weeks of injection to determine whether the infant mice have autism behavior. In the present application, the three most significant behaviors of autism patients: poor or less social ability (social interaction), obvious repetitive stereotyped behavior and anxiety behavior can be detected by the open field experiment.

[0039] In order to further illustrate the present application, a method for inducing a rat autism model by using a neurotoxin is described in detail in the present application in combination with examples, but they cannot be understood as limiting the scope of protection of the present application.

[0040] In example 1 and example 2 of the present application, the female mice are raised according to the mode shown in Figure 1, and at most four mice are placed in each cage with sufficient food and water. When the drug is administered, the female mice are administered according to the mode shown in Figure 2. The injection is selected at a loose subcutaneous tissue site, and the subcutaneous injection is performed on both sides of the abdomen. The drug solution is pushed to make the injection site protrude, and when the needle is pulled out, the needle puncture site is pinched with fingers to prevent the drug solution from leaking out. Sterile gloves and sterile protective clothing are worn, and the operation is performed in a feeding laboratory.

[0041] In example 1, the open field test of the test infant mice is performed according to the mode of the open field test of the infant mice shown in Figure 3. The open field reaction box is 30-40 cm high and 100 cm long at the bottom, and the inner wall is black. A digital camera is placed 2 m above the open field, and the field of view needs to cover the entire inside of the open field. After the data recorded by the digital camera is processed by software, the motion trajectory heat map of the infant mice is generated.

[0042] Example 1

[0043] Establishment of a neonatal infant autism model by administering a drug to female mice

[0044] 1.1. Requirement for adult female mouse strain

[0045] Wistar adult female rats (manufacturer: Sperof Bio-Technology Co., Ltd. (Beijing)) 16, divided into two groups: low dose group, high dose group. In the low dose group, 8 rats are marked as S1, S2, S3, S4, S5, S6, S7, S8, and in the high dose group, 8 rats are marked as S9, S10, S11, S12, S13, S14, S15, S16.

[0046] 1.2. Preparation of reagents

[0047] BMAA solution preparation: 50 mg BMAA (manufacturer: Beijing Ino Kai Technology Co., Ltd., batch number: 150053) lyophilized powder was added to 7.3 mL of PBS solution (concentration: 0.01 M, manufacturer: BI, batch number: 2246306) to prepare a 6.9 mg / mL BMAA solution I. 0.33 mL of the BMAA solution I was taken out and added to 4.67 mL of PBS to dilute it to a 0.46 mg / mL BMAA solution II.

[0048] 1.3. Dosing operation of female mice

[0049] Toxicant administration to female mice in the test group: the low-dose group was subcutaneously injected with the prepared BMAA solution II at 1 mg / Kg, and the high-dose group was subcutaneously injected with the prepared BMAA solution I at 15 mg / Kg.

[0050] After subcutaneous administration, the female mice were gently placed back into the cage and observed for 15 min for their reactions and for 5 h for any death, and the death conditions were recorded. The mice were raised in an environment with adequate light and air at room temperature of 26±3°C, and were given sufficient standard feed and tap water.

[0051] 1.4. Control group test

[0052] The adult male and female mice were caged together to observe the pregnancy and birth of the female mice and the growth of the pups.

[0053] 1.5. Open field test

[0054] Behavioral tests were started 35 days after the birth of the pups in the test and control groups. The three most notable behaviors of autism patients are poor or less social ability (social interaction), obvious repetitive stereotyped behavior, and anxiety behavior. The behavior of the pups was observed, and the open field test was used to reflect the autism behavior of the pups.

[0055] (1) Adaptation stage: the tested pup was gently placed into the device (material: acrylic plate, length: 100 cm x width: 100 cm x height: 40 cm) and allowed to freely move for 10 min.

[0056] (2) Test stage: after the tested pup was adapted, the number of standing and wall climbing of the tested pup in the next 10 min was recorded, and the defecation and urination of the tested pup during the test stage was also recorded. The standing and wall climbing criteria were that both front limbs were off the ground and maintained for 2 s or more. After the test, the tested pup was gently taken out and placed back into the cage, and then the device was wiped with 75% alcohol solution. After drying, the next pup was tested.

[0057] 1.6. Analysis of open field test results

[0058] Fig. 4, Fig. 5 and Fig. 6 are the motion trajectory heat maps of the pups in the high-dose group, the low-dose group and the control group, respectively. The heat maps show that the activity range of the pups in the high-dose group is the smallest, followed by the low-dose group and the control group. The test results show that the higher the dose of BMAA given to the mother mouse, the greater the impact on the behavior of the pups.

[0059] In this batch of modeling, the success rate of the low-dose group is about 40%, and the success rate of the high-dose group is about 60%. A total of 10 mice were tested in the high-dose group, 10 mice were tested in the low-dose group, and 6 mice were tested in the control group.

[0060] Example 2

[0061] Establishment of a mouse autism model by administering BMAA to newborn pups

[0062] 2.1. Requirements for adult female mouse strains

[0063] Wistar adult female rats (manufacturer: Sperof Bio-Technology Co., Ltd. (Beijing)).

[0064] 2.2. Preparation of reagents

[0065] Preparation of BMAA solution: 40 mg of BMAA (manufacturer: Beijing Inokai Technology Co., Ltd., batch number: 150053) lyophilized powder was added to 4.0 mL of PBS solution (concentration: 0.01 M, manufacturer: Biological Industries (BI), batch number: 2246306) to prepare a 10 mg / mL BMAA solution.

[0066] 2.3. Pup administration

[0067] The adult male and female mice were caged together, and the pregnancy and birth of the female mice were observed, and the growth of the pups was observed. After 25 days of birth, the pups were divided into two groups: a control group and a test group. The control group was not injected with BMAA solution; the test group of pups was calculated according to the weight of the pups and the dose of BMAA toxin administered to the pups, and the test group of pups was injected with BMAA toxin subcutaneously at a dose of 50 mg / kg body weight. After subcutaneous administration, the pups were gently returned to the cage and observed for 15 minutes for any reaction and for 5 hours for any death, and the death was recorded. The pups were raised in a room with adequate light and air at a normal temperature of 26±3°C.

[0068] 2.4. Open field test

[0069] The behavior test was started 35 days after the birth of the test group and the control group of pups. The three most notable behaviors of autism patients are poor or less social ability (social interaction), obvious repetitive stereotyped behavior, and anxiety behavior. Observation of the behavior of the pups can reflect the autism behavior of the pups using the open field test.

[0070] (1) Adaptation stage: the tested infant mice were gently put into the device (material: acrylic plate, length: 100 cm x width: 100 cm x height: 40 cm) and allowed to freely move for 10 min.

[0071] (2) Test stage: after the tested infant mice were adapted, the number of standing and climbing of the tested infant mice in the next 10 min was recorded, and the defecation and urination of the tested infant mice in the test stage was recorded. The standing and climbing standard: both forelimbs were off the ground and maintained for 2 s or more. After the test, the tested infant mice were gently taken out and put back into the cage, then the device was wiped with 75% alcohol solution, and the next infant mouse was tested after the device was dried.

[0072] 2.5. Open field test result analysis

[0073] FIGS. 7 and 8 are respectively the motion trajectory heat map of the control group and the test group infant mice. The heat map shows that the activity range of the test group infant mice is smaller than that of the control group. The test results show that BMAA injection directly to the newborn infant mice also has a significant impact on the behavior of the infant mice.

[0074] The success rate of this batch of modeling is about 40%, and the test mice in the test group are 6, and the test mice in the control group are 6.

[0075] Example 1 and Example 2 are respectively injecting BMAA to the mother mice and injecting BMAA to the newborn infant mice, and the open field test results show that the behavior of the newborn infant mice is abnormal after the two ways of administration, and the autism behavior is shown. Therefore, the test proves that the influence of BMAA on infant mice can be through mother-to-infant transmission, or can be directly on the infant mice after birth, which is not possessed by other autism models at present. The above examples achieve the purpose of constructing the autism model of newborn infant mice by injecting BMAA.

[0076] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. Use of β-N-methylamino-L-alanine in the preparation of an autism animal model.

2. Use according to claim 1, characterized in that, The autism animal model comprises an autism rat model.

3. Use according to claim 2, characterized in that, The rat strain used in the autism rat model comprises Wistar rats, SD rats, F344 rats, Lewis rats, SHR rats or WKY rats.

4. A method for inducing a rat juvenile autism model using a neurotoxin, characterized by, The autism rat model comprises the following steps: injecting β-N-methylamino-L-alanine into a female rat, and then mating the female rat with a male rat, and then giving birth to a baby rat.

5. The method of claim 4, wherein, The injection comprises injecting a PBS solution of β-N-methylamino-L-alanine.

6. The method of claim 5, wherein, The injection amount of the injection is 1-1000 mg / kg of the body weight of the rat.

7. The method of claim 4 wherein, The injection method comprises subcutaneous injection.

8. The method of claim 7, wherein, The subcutaneous injection is performed once.

9. The method of claim 4 wherein, After the baby rat is born, the following step is further included: using an open field test to verify whether the baby rat has autism behavior.

10. The preparation method according to claim 9, characterized in that, The open field test is performed 3-6 weeks after the baby rat is born.

11. A method for inducing a rat pup model of autism with a neurotoxin, comprising the steps of, The autism rat model comprises the following steps: injecting β-N-methylamino-L-alanine into a baby rat that is born normally, and then using an open field test to verify whether the baby rat has autism behavior.

12. The method of claim 11, wherein, The injection comprises injecting a PBS solution of β-N-methylamino-L-alanine.

13. The method of claim 12, wherein, The injection amount of the injection is 1-200 mg / kg of the body weight of the baby rat.

14. The method of claim 11, wherein, The injection method comprises subcutaneous injection.

15. The method of claim 14, wherein, The subcutaneous injection is performed once.

16. The method of claim 11, wherein, After the injection, the following step is further included: using an open field test to verify whether the baby rat has autism behavior.

17. The method of claim 16, wherein, The open field test is performed 1-2 weeks after the injection.

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