A method of constructing a model of social novelty deficit by maternal rat catechol exposure

Abstract

The application discloses a method for constructing a social novelty defect model through catechol exposure of pregnant mice, and belongs to the technical field of disease animal models. The method comprises the following steps: performing catechol exposure treatment on the pregnant mice, feeding the pregnant mice until childbirth, and waiting for the obtained offspring mice to grow to a required age, so as to obtain the social novelty defect model. The method is capable of precisely matching a key time window of embryonic neural development, and adopts a single catechol exposure mode, so that the method is simple to operate, has a high model formation rate, and has a high survival rate of the mother mice and the offspring. In addition, the induced social novelty defect model of the offspring mice is stable in phenotype and strong in specificity, can simulate the cross-generation influence of maternal pregnancy environment exposure on abnormal social functions of offspring young, and is suitable for mechanism research, drug screening and diagnosis marker development of social impairment diseases such as autism.

Description

Technical Field

[0001] This invention relates to the field of animal model technology for diseases, specifically to a method for constructing an animal model in which offspring exhibit social novelty deficit behavior induced by catechol exposure during mouse pregnancy, and the application of this model in screening drugs for the prevention or treatment of social disorders. Background Technology

[0002] Social Novelty Deficit (SND) is a highly specific core social phenotype in Autism Spectrum Disorder (ASD). Distinguished from typical "social avoidance / decreased social interest," SND refers to an individual's lack of normal exploratory motivation, preference, and approach behavior towards unfamiliar social objects or novel social interaction patterns in social situations. Simultaneously, the individual is unable to adjust their social behavior in response to novel social cues, manifesting as a preference for familiar social objects, avoidance of unfamiliar social interactions, rigid and inflexible social behavior, and inability to adapt to new social situations. This phenotype is the core subtype of "persistent social interaction deficit" in ASD according to DSM-5 and is one of the most typical social characteristics of individuals with ASD. Social novelty deficit is a hallmark social phenotype that distinguishes ASD from other neurodevelopmental disorders (ADHD, intellectual disability) and mental illnesses. However, current research on social novelty deficit is extremely insufficient. Existing ASD models can only generalize and simulate abnormal social behavior, failing to accurately replicate the specific phenotype and pathological mechanisms of social novelty deficit. Furthermore, the occurrence and development of this phenotype are directly linked to the gene-environment interaction etiology and damage to core neural circuits in ASD. In addition to autism, diseases such as schizophrenia, depression, and anxiety disorders are often accompanied by social dysfunction. For example, patients with depression may exhibit social withdrawal and lack of interest in social activities, while patients with anxiety disorders are prone to fear and avoidance behaviors in social situations. These diseases also lack targeted diagnostic and treatment methods. Therefore, establishing reliable animal models of social deficit can provide an experimental platform for research on social impairments related to diseases such as ASD.

[0003] Many neurodevelopmental mental disorders, such as autism, originate in the critical stages of neural development from the embryonic period to early childhood. However, existing models are mostly constructed using adult mice, whose neural development is largely complete. This leads to significant discrepancies between research results obtained from adult mouse models and clinical realities. The pregnant mouse drug induction method is an important means of simulating social deficits in offspring caused by abnormal neural development during the embryonic period. This method closely reflects the pathophysiological characteristics of human fetuses influenced by the maternal environment and has greater clinical translational value compared to adult mouse models and gene-editing models. However, existing pregnant mouse drug induction models suffer from problems such as unclear administration methods and ambiguous administration times, leading to cumbersome procedures, physiological stress in maternal mice, and low survival rates in pups.

[0004] Catechol, chemically known as catechol or pyroquinone, with the molecular formula C6H4(OH)2, is widely distributed in various plants and their derivatives in nature. As an important fine chemical raw material, catechin has wide applications in pesticides, pharmaceuticals, fragrances, dyes, photosensitive materials, and the rubber industry. It is commonly used as a rubber hardener, electroplating additive, skin disinfectant, hair dye, photographic developer, and antioxidant. Currently, there are no reports of using catechin in establishing models related to social deficits. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a method for constructing a social novelty defect model by exposure of pregnant mice to catechol, aiming to solve the technical problems such as existing models not conforming to the pathological characteristics of human fetal neural development affected by maternal environmental exposure, emphasizing physiological stress in pregnant mice, and low survival rate of pups.

[0006] To achieve the above objectives, the present invention specifically adopts the following technical solution: A method for constructing a social novelty defect model by exposing pregnant mice to catechols involves: exposing pregnant mice to catechols, feeding the pregnant mice until delivery, and then using offspring mice that have reached the required age to form a social novelty defect model.

[0007] Preferably, the above method specifically includes the following steps: S1. Select adult male and female mice and acclimatize them, then mate the male and female mice together in the same cage. S2. Pregnant mice were given a single catechol exposure when they were 12-13 days pregnant. S3. The model of social novelty defect is to raise pregnant mice until delivery and then let them nurse their offspring mice. After weaning, the offspring mice are raised to the required age.

[0008] More preferably, in step S2 of the above method, catechol exposure refers to injecting a catechol solution into a pregnant mouse via intraperitoneal injection, and the injection dose of catechol is 50 mg / kg (catechol dosage / mouse body weight). The catechol solution can be prepared using physiological saline.

[0009] Preferably, in the above method, the obtained social novelty defect model can be subjected to phenotypic verification and specificity detection through the following steps: performing social novelty defect phenotypic verification using a three-box social experiment; and performing specificity verification using at least one of the following experiments: new object recognition experiment, open field experiment, elevated cross maze experiment, suspended tail experiment, forced swimming experiment, and marble burial experiment.

[0010] The mouse social novelty deficiency behavior model constructed in this invention can be used in at least the following scenarios: 1) Screening or evaluating drugs that intervene in social deficits of ASD; 2) Investigate the pathogenesis of ASD, especially the neural circuits and molecular mechanisms related to social deficits; 3) Screening for diagnostic biomarkers associated with ASD’s social novelty deficit.

[0011] Compared with the prior art, the beneficial technical effects of the present invention include: (1) This invention uses a pregnant mouse drug induction method to conduct a single exposure to catechols on the 12th-13th day of pregnancy in wild-type female mice. This time is at the critical stage of matching embryonic neural development, which is more in line with the pathological characteristics of human fetal neural development affected by maternal environmental exposure. The model has high clinical translational value and provides an effective tool for evaluating neurodevelopmental toxicity and screening anti-social disorder drugs.

[0012] (2) The present invention uses a single intraperitoneal injection method, which is simple to operate and takes less time compared with the traditional multiple exposure method. It can significantly reduce the physiological stress of the mother mouse and improve the conception rate of the mother mouse and the survival rate of the offspring mice.

[0013] (3) The model constructed in this invention has strong specificity and good stability. The offspring mice in the model group only showed social novelty deficiency, and their memory ability, motor ability, anxiety level, depression level, etc. were all normal, without other non-specific interference.

[0014] (4) The experimental phenotypes constructed by the present invention are stable and highly reproducible, which can effectively reduce research costs and improve research efficiency. Attached Figure Description

[0015] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0016] Figure 1 This is a flowchart illustrating the construction of a social novelty defect model using catechol exposure in pregnant mice, as described in this invention. Figure 2 The diagram shows the results of the three-box social experiment for the offspring mouse social novelty defect model obtained in the example. A is a schematic diagram of the three-box social experiment; B is a comparison of the exploration time of each group of mice in the left and right empty box areas during the habituation stage; C is a comparison of the exploration time of each group of mice to unfamiliar mice and empty cages in the social ability test; and D is a comparison of the exploration time of each group of mice to familiar mice and unfamiliar mice in the social novelty test. Figure 3The figure shows the experimental results of the new object recognition of the offspring mouse social novelty defect model obtained in the example. A is a schematic diagram of the new object recognition experiment, B is a comparison of the time of each group of mice in the exploration and memorization of objects, and C is a comparison of the exploration time of each group of mice for familiar objects and new objects. Figure 4 The diagram shows the open field experiment results of the offspring mouse social novelty defect model obtained in the example, where A is a schematic diagram of the open field experiment, B is the comparison result of the total movement distance of the mice, and C is the comparison result of the movement distance of the mice in the central region. Figure 5 The diagram shows the results of the elevated cross maze experiment for the offspring mouse social novelty defect model obtained in the examples. A is a schematic diagram of the elevated cross maze experiment, B is the comparison result of the open arm dwell time, and C is the comparison result of the closed arm dwell time. Figure 6 The image shows the results of the marble burial experiment on the offspring mouse social novelty defect model obtained in the example, where A is a schematic diagram of the marble burial experiment and B is a comparison of the number of buried marble balls. Figure 7 The diagram shows the tail suspension test results of the offspring mouse social novelty defect model obtained in the example, where A is a schematic diagram of the tail suspension test and B is a comparison result of the immobility time ratio. Figure 8 The diagram shows the results of the forced swimming experiment of the offspring mouse social novelty deficiency model obtained in the example, where A is a schematic diagram of the forced swimming experiment and B is a comparison of the proportion of immobility time. Explanation of reference numerals in the attached figures: P ≤0.0001, P ≤0.001, P ≤0.01, P ≤0.05, ns indicates no significance. Detailed Implementation

[0017] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0019] Social novelty deficit is a core phenotype of various neurodevelopmental mental disorders, including autism spectrum disorder. These disorders often originate in critical stages of neurodevelopment from the embryonic period to early childhood. However, existing models are mostly constructed using adult animals (such as adult mice), resulting in low clinical translation value. Although the pregnant mouse drug induction method can effectively simulate social deficits in offspring caused by abnormal neurodevelopment during the embryonic period, the use of drugs such as valproic acid, clozapine, and alcohol to induce social deficits in offspring requires strict control of the administration time and dosage, which is difficult to implement and can lead to problems such as pregnant mouse abortion, newborn mouse mortality, and low model success rate. To address the technical problems existing in constructing a social novelty deficit model based on the pregnant mouse drug induction method, this invention provides a method for constructing a social novelty deficit model through catechol exposure in pregnant mice. This method is simple to operate, has a high model success rate, and the resulting social novelty deficit model has strong phenotypic specificity, which can meet the needs of mechanism research on social impairment caused by environmental exposure during the embryonic period.

[0020] like Figure 1 As shown, the method for constructing a social novelty defect model through catechol exposure in pregnant mice provided in this embodiment of the invention includes the following steps: (1) Select male and female mice and acclimatize them, then put the male and female mice together for mating; (2) A single catechol exposure was given to pregnant mice when they were 12-13 days pregnant; (3) Feeding pregnant mice until delivery and feeding offspring mice, and then feeding offspring mice to the required age after weaning, is the social novelty defect model.

[0021] In a preferred embodiment of the present invention, step S2 specifically involves: preparing a catechol solution using physiological saline as a solvent, and injecting the catechol solution into the pregnant mouse intraperitoneally when the mouse is 12-13 days pregnant, with the catechol injection dose being 50 mg / kg.

[0022] In a preferred embodiment of the present invention, in step (3), the offspring mice are raised to 6 weeks of age. At this time, the offspring mice are used as a social novelty defect model with stable phenotype and strong specificity. Moreover, they are more in line with the onset stage of human social disorder (early childhood and adolescence) and can simulate the pathophysiological characteristics of abnormal social function in human early childhood.

[0023] In a preferred embodiment of the present invention, the rearing conditions for mice include: a 12-hour light-12-hour dark cycle (07:00-19:00), a temperature of 20-22°C, a humidity of 45-60%, 3-5 mice per cage, and regular replacement and disinfection of feed and bedding.

[0024] In a preferred embodiment of the present invention, the method for constructing a social novelty defect model further includes the following steps: The social novelty defect phenotype was validated using a three-box social experiment. The determination method was as follows: if the offspring mice of pregnant mice exposed to catechol showed no significant difference in exploration time between familiar mice and unfamiliar mice in the social novelty test, while the control offspring mice of pregnant mice not exposed to catechol showed a significantly longer exploration time for unfamiliar mice than for familiar mice, then the social novelty defect model could be determined to have been successfully constructed.

[0025] At least one of the following experiments was used for specific verification: novel object recognition experiment, open field experiment, elevated cross maze experiment, marble burial experiment, tail suspension experiment, and forced swimming experiment. This was to verify that the offspring mice showed no abnormalities in short-term memory, anxiety, or depression, and that there were no other non-specific interferences, thus ensuring that the phenotype was a social novelty-specific defect.

[0026] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0027] Example 1 This example provides a method for constructing a social novelty defect model through catechol exposure in pregnant mice, including the following steps: (1) Selection and adaptive feeding of experimental animals.

[0028] Eight-week-old male C57BL / 6J mice (weighing 20±1 g) and eight-week-old female C57BL / 6J mice (weighing 18±1 g) were selected as experimental parents. All mice had no history of neuropsychiatric diseases, physical diseases, or drug exposure.

[0029] Male and female parent mice were housed separately in an environment with a 12-hour light-dark cycle (lighting time from 7:00 to 19:00 daily), a temperature of 20-22℃, and a humidity of 45-60%, with 3-5 mice per cage. Sufficient feed and water were provided for 7 days for acclimatization.

[0030] (2) Preparation of catechin solution.

[0031] Take 20 mg of catechin (purity ≥99%), add sterile physiological saline and stir to dissolve thoroughly, then bring the volume to 3 mL to obtain a catechin solution (prepare and use immediately; discard any unused catechin solution promptly and do not reuse).

[0032] Previous experiments have shown that 50 mg / kg is the maximum dose that mice can safely tolerate via intraperitoneal injection. Therefore, in this case, the daily dosage for mice is 50 mg / kg (catechin weight / mouse body weight), and a 20 g mouse would require an injection of 0.15 mL of the above-mentioned catechol solution.

[0033] (3) Catechol exposure treatment in pregnant rats.

[0034] After acclimatization, female and male mice were mated at 6 PM in a 1:2 ratio. Vaginal plugs were checked at 8 AM daily, and mice with detected vaginal plugs were considered to be on day 0.5 of pregnancy. On day 12.5 of gestation, the pregnant mice were randomly divided into experimental and control groups. The experimental group received a single intraperitoneal injection of the catechol solution prepared in step (2) at a dose of 50 mg / kg, while the control group received an equal volume of physiological saline at the same time. The pregnant mice were fed normally until delivery, and the feeding environment was consistent with step (1).

[0035] (4) Feeding offspring mice.

[0036] The day the pregnant mouse gives birth is recorded as day 0 after birth. After 21 days of nursing, the offspring mice are weaned and separated by sex and group. The feeding environment is the same as in step (1), and they are fed normally until they are 6 weeks old.

[0037] Example 2 This example uses offspring mice obtained in Example 1 to test their effectiveness as a social novelty defect model. The specific experiments and results analysis are as follows: Six-week-old offspring mice were selected, and according to the grouping in Example 1, the resulting offspring mice were designated as the model group offspring (CAT, n=12) and the control group offspring (NS, n=10), respectively; each group of mice contained both female and male mice, and there was no significant difference in body weight between the two groups. P >0.05), to ensure the reliability of the experimental results.

[0038] (1) Phenotypic verification was performed using a three-box social experiment.

[0039] A 90×30×30 cm acrylic three-box assembly (divided into left, middle, and right areas, such as...) is used. Figure 2 As shown in A), according to "habituation" Social skills test The "Social Novelty Test" was conducted in three phases: Habituation phase: Place the mouse in the central area and allow it to explore freely for 10 minutes; Social skills test: An unfamiliar mouse (restrained in a cylindrical cage, denoted as S1) was placed in one area, and an empty restraint cage (denoted as S2) was placed in the other area. The mouse was tested for 10 minutes. Social novelty test: The familiar S1 is retained, and the other empty cage is replaced with another new unfamiliar mouse (denoted as new S2). The test mice are tested for 10 minutes. After each stage, the device was wiped with 75% alcohol to remove the odor. The exploration time and movement trajectory of the mice within a 7cm range of the restraint cage were recorded using VisuTrack software.

[0040] The criteria for the above three-box social experiment are as follows: if there is no statistically significant difference in the exploration time of S1 and new S2 in the drug-treated group during the social novelty test (p>0.05), while the exploration time of new S2 in the control group is significantly longer than that of S1 (p<0.05), then the social novelty behavior defect model is considered to have been successfully constructed.

[0041] Experimental results are as follows Figure 2 As shown in B-2D, during the social novelty test, the offspring mice in the drug-treated group showed no statistically significant difference in exploration time between familiar and unfamiliar mice, indicating no preference for new social objects. In contrast, the offspring mice in the control group spent significantly longer exploring unfamiliar mice than familiar mice, exhibiting a normal social novelty preference. This demonstrates that the juvenile social novelty behavior deficiency model constructed in this invention was successfully established.

[0042] (2) Specificity verification.

[0043] After the three-box social experiment was completed, the two groups of offspring mice were subjected to new object recognition experiment, open field experiment, elevated cross maze experiment, marble burial experiment, tail suspension experiment and forced swimming experiment in sequence to eliminate interference factors. All experiments were completed within one week after the three-box social experiment.

[0044] ① New object recognition experiment.

[0045] Because cognitive deficits in mice can severely interfere with their assessment of social novelty deficits, the new object recognition experiment can assess whether a mouse's cognitive level and short-term memory have changed. The new object recognition experiment consists of two phases: the first phase is a training phase, aimed at training mice to remember objects; the second phase is a new object recognition test phase, aimed at observing and recording the time mice spend exploring familiar and new objects respectively. Mice with normal short-term memory will spend more time exploring new objects. Figure 3 As shown in A, the experiment can be conducted in a 45×45×45 cm open field device. Two objects are placed side by side 15 cm away from the wall, with a distance of 15 cm between the two objects. The experiment uses low light intensity. After each stage, the device is wiped with 75% alcohol to remove the odor. The exploration time and movement trajectory of the mice can be recorded using VisuTrack software.

[0046] Experimental results are as follows Figure 3As shown in B-3C, the proportion of time spent exploring new objects in the offspring mice of the drug-treated group was not statistically significantly different from that in the control group. This indicates that the short-term memory and cognitive levels of the model mice constructed in this study were not altered, and their social novelty deficit was not caused by reduced cognitive ability or memory impairment.

[0047] ② Open field experiment, elevated cross maze experiment and marble burial experiment.

[0048] like Figure 4 As shown in Figure A, the open field experiment was conducted as follows: a 45×45×45 cm open field box was used, and the time the mouse spent in the central area and the total distance traveled were recorded within 10 minutes. The entire experiment was recorded using an animal behavior analysis system. The results are as follows: Figure 4 As shown in B-4C, there was no significant difference in the exploration distance of mice in the central region between the drug-treated progeny group and the control progeny group.

[0049] like Figure 5 As shown in Figure A, the elevated cross maze experiment specifically involved recording the time the mouse spent in the open arm within 5 minutes. The results are as follows: Figure 5 As shown in B-5C, there was no significant difference in the dwell time of mice in the drug-treated progeny group and the control progeny group on the open and closed arms.

[0050] like Figure 6 As shown in Figure A, the marble burial experiment specifically involved recording the number of marbles buried by mice within 30 minutes. The results are as follows: Figure 6 As shown in Figure B, there was no significant difference in the number of marbles buried by mice in the drug-treated progeny group and the control progeny group.

[0051] The open field test, the elevated cross maze test, and the marble burial test can assess the anxiety level of mice. The above results show that the childhood social novelty behavior deficit model constructed in this invention has no anxiety-like behavior.

[0052] ③ Tail suspension test and forced swimming test.

[0053] like Figure 7 As shown in Figure A, the forced swimming experiment specifically involved recording the proportion of time the mice spent floating in the pool within a 6-minute period. The results are as follows: Figure 7 As shown in B, there was no significant difference in immobility time between the drug-treated progeny group and the control progeny group.

[0054] like Figure 8 As shown in Figure A, the tail suspension experiment specifically involved recording the proportion of time the mouse remained immobile during suspension over a 6-minute period. The results are as follows: Figure 8 As shown in B, there was no significant difference in immobility time between the drug-treated progeny group and the control progeny group.

[0055] The tail suspension test and forced swimming test can assess depressive-like behavior in mice. The above results indicate that the childhood social novelty behavior deficit model constructed in this invention does not exhibit depressive-like behavior.

[0056] In summary, the offspring mice prepared by the present invention through exposure to catechols during pregnancy can be used as a model of social novelty deficiency in juveniles. The phenotype of this model is stable and highly specific, that is, it only shows social novelty deficiency, with no abnormalities in memory, motor ability, anxiety level, depression level, etc., and no other non-contaminating factors.

[0057] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for constructing a social novelty defect model through exposure to catechols in pregnant mice, characterized in that, Includes the following steps: S1. Select adult male and female mice, and after acclimatization, mate the male and female mice together in the same cage. S2. When the pregnant mice are 12-13 days pregnant, they are given a single catechol exposure treatment; the catechol exposure is to inject catechol solution into the pregnant mice intraperitoneally, and the injection dose of catechol is 50 mg / kg. S3. Feeding pregnant mice until delivery, allowing them to nurse offspring mice, and then feeding the offspring mice to the required age after weaning, constitutes the social novelty defect model.

2. The method according to claim 1, characterized in that, Catechol injection solution was prepared using physiological saline.

3. The method according to claim 1, characterized in that, The offspring mice can be used as a social novelty defect model when they reach 6-7 weeks of age.

4. The method according to claim 1, characterized in that, It also includes the following steps: The obtained social novelty defect model was phenotypically validated using a three-box social experiment.

5. The method according to claim 1, characterized in that, It also includes the following steps: The obtained social novelty defect model was specifically verified by using at least one of the following experiments: novel object recognition experiment, open field experiment, elevated cross maze experiment, suspended tail experiment, forced swimming experiment, and marble burial experiment.

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