Method for establishing and verifying a hydrogen peroxide-induced mouse autism model

Abstract

This invention discloses a method for establishing and validating a hydrogen peroxide-induced mouse autism model, relating to the field of medical biotechnology. The method for establishing the hydrogen peroxide-induced mouse autism model includes the following steps: injecting the hydrogen peroxide solution into the peritoneal cavity of pregnant mice to induce hydrogen peroxide, resulting in offspring mice with hydrogen peroxide induction, i.e., the mouse autism model. This invention uses near-peritoneal injection of hydrogen peroxide during the gestational period of the mother mouse, inducing oxidative stress in both the pregnant mother mouse and the offspring embryo through hydrogen peroxide alone, without causing a significant immune response in the mother mouse. Therefore, an oxidative stress-specifically induced ASD mouse model is formed. The resulting ASD offspring mice exhibit significant ASD phenotypes but do not show obvious motor impairment.

Description

Technical Field

[0001] This invention relates to the field of medical biotechnology, and in particular to a method for establishing and validating a hydrogen peroxide-induced mouse autism model. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication, repetitive and restricted behavioral patterns, and narrow range of interests or activities. To study the pathological mechanisms of ASD and develop treatments, animal models of autism, particularly mouse models, are typically constructed to mimic the symptoms and biological characteristics of human ASD.

[0003] Currently, common ASD mouse models mainly include two types: (1) induction models represented by maternal immune activation (MIA) in offspring mice, and (2) transgenic models represented by BTBR strain mice and specific transgenic strain mice. Among them, although the MIA inflammation-induced model is consistent with the clinical occurrence of ASD in terms of disease course simulation and ASD phenotype, this model not only introduces exogenous immunogenic substances such as lipopolysaccharide (LPS) and polyinosinic acid (Poly(I:C)), but also causes widespread immune activation and release of inflammatory factors, as well as activation of various inflammatory pathways. At the molecular level, it is interfered with by complex factors such as the toxicity of exogenous substances themselves, immune damage, and oxidative stress, which to some extent hinders the screening of key pathways leading to autism disorder. However, the neurological phenotypes of BTBR mice and transgenic mice do not necessarily lead to typical autism disorder. They are often accompanied by more severe cognitive and motor impairments. Furthermore, at the molecular level, changes in specific genes may cause changes in a wider range of signaling pathways closely related to those genes. These changes may interfere with or dilute the alterations in key neural pathways that contribute to the development of autism disorder, which is not conducive to molecular-level research.

[0004] Therefore, there is still a lack of universal animal models for studying ASD. Summary of the Invention

[0005] The main objective of this invention is to propose a method for establishing and validating a hydrogen peroxide-induced mouse autism model, aiming to solve the problem of the lack of a universal animal model for ASD research in the prior art.

[0006] To achieve the above objectives, this invention proposes a method for establishing a hydrogen peroxide-induced mouse autism model, comprising the following steps:

[0007] Hydrogen peroxide solution was injected into the peritoneal cavity of pregnant mice to induce hydrogen peroxide, resulting in offspring mice that were induced by hydrogen peroxide, which are mouse autism models.

[0008] In one embodiment, the method for establishing the hydrogen peroxide-induced mouse autism model includes:

[0009] The hydrogen peroxide solution was injected intraperitoneally into pregnant mice on day 12.5 of embryonic development to induce hydrogen peroxide, resulting in offspring mice induced by hydrogen peroxide, which are mouse autism models.

[0010] In one embodiment, the amount of hydrogen peroxide injected into the peritoneal cavity of the pregnant mouse is 180–220 μmol / kg.

[0011] In one embodiment, the hydrogen peroxide induction time is 6 to 72 hours.

[0012] The present invention also provides a method for verifying a mouse autism model, wherein the method for verifying a mouse autism model is used to verify the mouse autism model obtained by the aforementioned method for establishing a hydrogen peroxide-induced mouse autism model;

[0013] The validation method for the mouse autism model includes the following steps:

[0014] To assess the motor abilities, stereotyped grooming behaviors, and social interaction abilities of a mouse model of autism;

[0015] To assess synaptic changes in a mouse model of autism;

[0016] To assess the expression of autism spectrum disorder-related pathogenic genes in a mouse autism model.

[0017] In one embodiment, the step of assessing brain synaptic changes in a mouse autism model includes:

[0018] Evaluation of brain synaptic histology in a mouse model of autism;

[0019] To evaluate brain synaptic markers in a mouse model of autism.

[0020] In one embodiment, the step of assessing the expression of autism spectrum disorder-related pathogenic genes in a mouse autism model includes:

[0021] To assess the expression of autism spectrum disorder-related pathogenic genes in a mouse model of autism;

[0022] To evaluate differentially expressed gene enrichment pathways in a mouse model of autism spectrum disorder.

[0023] In one embodiment, the brain synaptic markers include at least one of Synaptophysin and Postsynapticdensity protein-95.

[0024] In one embodiment, the autism spectrum disorder-related pathogenic genes include at least one of Adcy5, Adora2a, Agtr2, Cep135, Cep290, Cx3cr1, Ddc, Drd1, Drd2, Ebf3, En2, Esrrb, Fhit, Gpr139, Htr7, Il16, Lmx1b, Nckap5, Nfe2l3, P2rx5, Patj, Pax5, Pcdha4, Pcdh9, Slc29a4, Slc6a3, Slc6a4, Ppp1r1b, Sh3rf2, Tph2, Ttn, and Wnt2.

[0025] In one embodiment, differentially expressed gene enrichment pathways in autism spectrum disorder include neurotransmitter-receptor interactions, dopaminergic neurons, retrosynaptic signaling, and oxidative phosphorylation.

[0026] In this invention, hydrogen peroxide is injected intraperitoneally during the gestation period of pregnant mice, inducing oxidative stress in both the pregnant mothers and their offspring embryos without causing a significant immune response in the mothers, unlike existing technologies. Furthermore, H2O2 decomposes into H2O2 and O2 after a redox reaction, neither of which is toxic or irritating to mice, thus creating an oxidative stress-specific induced ASD mouse model. The ASD offspring mice obtained by this invention exhibit a significant ASD phenotype but do not show obvious motor impairment, highlighting the typicality of the ASD characteristics in this mouse model. This invention achieves the effect of embryonic oxidative stress through intraperitoneal injection of hydrogen peroxide only during the gestation period of the mother, is convenient and inexpensive, and does not cause a significant immune inflammatory response in the mothers, effectively reducing the suffering of the experimental mice and greatly benefiting the protection and ethical implementation of experimental animal welfare. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0028] Figure 1The graph shows the results of serum CRP immune index detection in female mice on days 0, 1, 2 and 3 after H2O2 injection in Example 2 of this invention.

[0029] Figure 2 Image (A) shows the CAT detection results in embryos derived from the first pair of H2O2-injected pregnant mice (H2O2) and uninjected control pregnant mice (CTR) in Example 2 of this invention. Figure 2 Image (B) shows the CAT detection results in embryos derived from the second pair of H2O2-injected pregnant mice (H2O2) and uninjected control pregnant mice (CTR) in Example 2 of the present invention.

[0030] Figure 3 (A) is a graph showing the open field experiment results of offspring mice induced by H2O2 during pregnancy in Example 3 of the present invention. Figure 3 (B) is a graph showing the results of the rotating rod experiment of offspring mice induced by H2O2 during pregnancy in Example 3 of the present invention;

[0031] Figure 4 (A) is a statistical chart showing the total time of grooming behavior in offspring mice induced by H2O2 during pregnancy in Example 4 of the present invention. Figure 4 (B) is a frequency statistical graph of grooming behavior in offspring mice induced by H2O2 during pregnancy in Example 4 of the present invention;

[0032] Figure 5 Image (A) is a schematic diagram of the three-box socialization experiment protocol for offspring mice induced by H2O2 during pregnancy in Example 5 of the present invention. Figure 5 (B) is a graph showing the results of the three-box social ability assessment of offspring mice induced by H2O2 during pregnancy in Example 5 of the present invention;

[0033] Figure 6 Image (A) shows the synaptic histological results of offspring mice induced by H2O2 during pregnancy in Example 6 of this invention. Figure 6 (B) is a graph showing the statistical results of the number of dendritic spines in offspring mice induced by H2O2 during pregnancy in Example 6 of the present invention;

[0034] Figure 7 The image shows the results of synaptic marker detection in offspring mice induced by H2O2 during pregnancy in Example 7 of this invention.

[0035] Figure 8 Image (A) is a volcano diagram of differentially expressed genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 of this invention. Figure 8 (B) is a statistical chart showing the results of downregulated (green) and upregulated (red) genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 of the present invention, which are clearly associated with the onset of autism.

[0036] Figure 9 The figure shows the GO enrichment analysis results of differentially expressed genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 of this invention.

[0037] Figure 10 The image shows the KEGG enrichment analysis results of differentially expressed genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 of this invention.

[0038] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication, repetitive and restricted behavioral patterns, and narrow range of interests or activities. To study the pathological mechanisms of ASD and develop treatments, animal models of autism, particularly mouse models, are typically constructed to mimic the symptoms and biological characteristics of human ASD.

[0041] Currently, common ASD mouse models mainly include two types: (1) induction models represented by maternal immune activation (MIA) in offspring mice, and (2) transgenic models represented by BTBR strain mice and specific transgenic strain mice. Among them, although the MIA inflammation-induced model is consistent with the clinical occurrence of ASD in terms of disease course simulation and ASD phenotype, this model not only introduces exogenous immunogenic substances such as lipopolysaccharide (LPS) and polyinosinic acid (Poly(I:C)), but also causes widespread immune activation and release of inflammatory factors, as well as activation of various inflammatory pathways. At the molecular level, it is interfered with by complex factors such as the toxicity of exogenous substances themselves, immune damage, and oxidative stress, which to some extent hinders the screening of key pathways leading to autism disorder. However, the neurological phenotypes of BTBR mice and transgenic mice do not necessarily lead to typical autism disorder. They are often accompanied by more severe cognitive and motor impairments. Furthermore, at the molecular level, changes in specific genes may cause changes in a wider range of signaling pathways closely related to those genes. These changes may interfere with or dilute the alterations in key neural pathways that contribute to the development of autism disorder, which is not conducive to molecular-level research.

[0042] Therefore, there is still a lack of universal animal models for studying ASD.

[0043] In view of this, the present invention provides a method for establishing a hydrogen peroxide-induced mouse autism model, comprising the following steps: injecting hydrogen peroxide solution into the peritoneal cavity of a pregnant mouse to induce hydrogen peroxide, thereby obtaining offspring mice induced by hydrogen peroxide, i.e., a mouse autism model.

[0044] In the technical solution of this invention, hydrogen peroxide is injected intraperitoneally into pregnant mice during pregnancy, inducing oxidative stress in both the pregnant mother mice and the offspring embryos through hydrogen peroxide alone, without causing a significant immune response in the mother mice, which differs from existing technologies. Furthermore, H2O2 decomposes into two products, H2O2 and O2, after a redox reaction. Neither product is toxic or irritating to mice, thus forming an oxidative stress-specific induced ASD mouse model. The ASD offspring mice obtained by this invention exhibit a significant ASD phenotype but do not show obvious motor impairment, highlighting the typicality of the ASD characteristics in the mouse model of this invention.

[0045] Because this invention achieves the effect of embryonic oxidative stress through intraperitoneal injection of hydrogen peroxide only during the pregnancy of the mother mouse, it is convenient to operate and low in cost; and it does not cause obvious immune inflammatory response in the mother mouse, effectively reducing the suffering of the experimental mouse, which is very beneficial to the protection and implementation of experimental animal welfare and ethics.

[0046] It should be noted that a wide range of substances are used to induce oxidative stress, many of which are biotoxic. These include heavy metals, antibiotics, anticancer drugs, and immunoantigens. While producing their own metabolic toxicity or inflammation, these substances also cause oxidative stress, and their exposure is often ambiguous. Therefore, these substances are not suitable for constructing ASD mouse models induced solely by oxidative stress. Furthermore, hydrogen peroxide is typically used only for clinical skin wound disinfection and is almost never used for in vivo administration.

[0047] In some embodiments of the present invention, the method for establishing the hydrogen peroxide-induced mouse autism model includes: injecting the hydrogen peroxide solution into the peritoneal cavity of a pregnant mouse on the 12.5th day of embryonic development to induce hydrogen peroxide, thereby obtaining offspring mice induced by hydrogen peroxide, i.e., mouse autism models.

[0048] In the technical solution of this invention, preferably, the pregnant mouse is injected intraperitoneally on day 12.5 of embryonic development. Selecting pregnant mice at this stage ensures a better influence on the nervous system development of the offspring mouse embryos, resulting in a faster mouse autism model.

[0049] In some embodiments of the present invention, the amount of hydrogen peroxide injected into the peritoneum of the pregnant mice is 180–220 μmol / kg. The amount of hydrogen peroxide injected into the peritoneum of the pregnant mice can be 180 μmol / kg, 200 μmol / kg, or 220 μmol / kg. Injection amounts within the above range can ensure a good oxidative stress induction effect, resulting in a more stable mouse autism model with a significant ASD phenotype.

[0050] It should be noted that the H2O2 injection time (E12.5) and injection dose (200 μmol / kg) involved in Example 1 represent a typical example of an implementation method, i.e. a preferred scheme. However, since the entire gestation period is a crucial stage of embryonic neural development, it can be inferred that injection at other time points, or the use of lower or higher injection doses, could theoretically also induce ASD in offspring mice.

[0051] In some embodiments of the present invention, the hydrogen peroxide induction time is 6–72 h. The hydrogen peroxide induction time can be 24 h, 48 h, 72 h or longer, and the induction time within the above range can ensure that the obtained mouse autism model is relatively stable and has a significant ASD phenotype.

[0052] It should be noted that in this invention, the injected hydrogen peroxide dose is a relatively low safe dose within the non-lethal dose range (4000 μmol / kg is the lethal dose). This injection dose and induction time will not cause death in pregnant mice because most of the injected H2O2 will rapidly decompose into O2 and H2O upon entering the body, and the body can completely absorb these gases, thus preventing direct death. At a lethal dose of 4000 μmol / kg, excessive oxygen generation will lead to a rapid increase in abdominal pressure, causing the pregnant mice to die from physical compression—a unique characteristic compared to other drugs.

[0053] This invention also provides a method for validating a mouse autism model. This method is used to verify the mouse autism model obtained by the aforementioned hydrogen peroxide-induced mouse autism model establishment method. The method includes the following steps: assessing the mouse autism model's motor abilities, stereotyped grooming behaviors, and social interaction abilities; assessing synaptic changes in the mouse autism model's brain; and assessing the expression of autism spectrum disorder-related pathogenic genes in the mouse autism model. These evaluation indicators can be used to quickly determine whether the offspring mice exhibit ASD characteristics.

[0054] In some embodiments of the present invention, the step of assessing synaptic changes in a mouse autism model includes: assessing the synaptic histology of the mouse autism model; and assessing synaptic biomarkers in the mouse autism model. By observing the synaptic histology and biomarkers, it is possible to quickly determine whether changes have occurred in the mouse brain synapses.

[0055] In some embodiments of the present invention, the step of assessing the expression of autism spectrum disorder-related pathogenic genes in a mouse autism model includes: assessing the expression of autism spectrum disorder-related pathogenic genes in the mouse autism model; and assessing the enrichment pathways of differentially expressed genes in the mouse autism model. By analyzing the expression of autism spectrum disorder-related pathogenic genes and the enrichment pathways of differentially expressed genes in the mouse autism model, it is possible to accurately determine whether a mouse is an ASD model and to quickly identify the specific pathway type that led to the establishment of the model.

[0056] In some embodiments of the present invention, the brain synaptic biomarkers include at least one of Synaptophysin and Postsynaptic density protein-95 (PSD95). These biomarkers can rapidly reflect changes in synapses.

[0057] In some embodiments of the present invention, the autism spectrum disorder-related pathogenic genes include adnylatecyclase 5 (Adcy5), adenosine A2a receptor (Adora2a), angiotensin II receptor type 2 (Agtr2), centrosomal protein 135 (Cep135), centrosomal protein 290 (Cep290), C-X3-C motif chemokine receptor 1 (Cx3cr1), dopa decarboxylase (Ddc), dopamine receptor D1 (Drd1), dopamine receptor D2 (Drd2), EBF transcription factor 3 (Ebf3), engrailed homeobox 2 (En2), estrogen related receptor beta (Esrrb), fragile histidine triad diadenosine triphosphatase (Fhit), G protein-coupled receptor 139 (Gpr139), 5-hydroxytryptamine receptor 7 (Htr7), and interleukin. 16(Il16), LIM homeobox transcription factor 1beta(Lmx1b), NCK associated protein5(Nckap5), NFE2 like bZIP transcription factor 3(Nfe2l3), purinergic receptorP2X 5(P2rx5), PATJ crumbs cell polarity complex component(Patj), paired box 5(Pax5), protocadherin alpha 4(Pcdha4), protocadherin 9(Pcdh9), solute carrier family 29member 4(Slc29a4), solute carrier family 6member 3(Slc6a3), solutecarrier family 6member 4(Slc6a4), protein phosphatase1regulatoryThe autism spectrum disorder-related pathogenic genes include at least one of the following: inhibitorsubunit 1B (Ppp1r1b), SH3 domain containing ring finger 2 (Sh3rf2), tryptophan hydroxylase 2 (Tph2), titin (Ttn), and Wnt family member 2 (Wnt2). These genes effectively reflect the successful establishment of the mouse autism model provided by this invention.

[0058] In some embodiments of the present invention, the differentially expressed gene enrichment pathways in autism spectrum disorder include neurotransmitter-receptor interactions, dopaminergic neurons, retrosynaptic signaling, and oxidative phosphorylation. These pathways illustrate the formation pathway of the mouse autism model establishment method provided by the present invention.

[0059] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0060] Example 1: Establishment of a mouse autism model

[0061] Mice were housed in an SPF (Specific Pathogen Free) environment. The onset of pregnancy was determined by cage bonding and examination of vaginal plugs. On day 12.5 of gestation (i.e., day 12.5 of embryonic development), pregnant mice were intraperitoneally injected with 200 μmol / kg of H2O2 (i.e., a 1:100 dilution of 30% H2O2 solution). 500 μL of this diluted H2O2 solution was injected intraperitoneally into each 25g pregnant mouse. The pregnant mice were then placed in a normal SPF environment for culture until the offspring were born. The offspring were then normally lactated, and the mouse autism model was obtained when the offspring reached adulthood (9 weeks of age).

[0062] Example 2: Assessment of oxidative stress and immune response in H2O2-induced embryos of mother and offspring rats during pregnancy.

[0063] The levels of C-reactive protein (CRP), an inflammatory marker, in the blood of pregnant mice at 0 days (6 h post-injection), 1 day (24 h post-injection), 2 days (48 h post-injection), and 3 days (72 h post-injection) following H2O2 injection were measured using enzyme-linked immunosorbent assay (ELISA). Twelve pregnant mice were injected with H2O2 on day E12.5. At each time point post-injection, three pregnant mice were completely anesthetized with afodin, and blood was collected by enucleation. The collected blood was allowed to stand at room temperature for 30 minutes before... Centrifuge at 2500×g for 10 minutes, collect the clear supernatant as serum, dilute with physiological saline at 1:400 for subsequent testing; prepare standard curve samples: dilute 10ng / mL mouse-derived CRP standard (purchased from Abcam, catalog number #222511) 50% into 7 gradients (10000pg / mL, 5000pg / mL, 2500pg / mL, 1250pg / mL, 625pg / mL, 156pg / mL), plus one tube of 0 concentration blank control. Add 50 μL of sample per well to a 96-well plate pre-coated with CRP antibody (horseradish peroxidase [HRP]), followed by 50 μL of CRP antibody (HRP) dilution buffer. Mix thoroughly and incubate at room temperature for 1 hour on a horizontal shaker at 400 rpm. Then, thoroughly wash the plate, removing as much excess liquid as possible. Next, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reagent for colorimetric reaction. Calculate the urinary protein content in the samples at different time points based on the standard curve. The detection results are as follows: Figure 1 As shown. In Figure 1 In the graph, the horizontal axis represents the detection time, from left to right: 6 hours (0), 1 day (1), 2 days (2), and 3 days (3) after injection; the vertical axis represents the serum CRP protein level obtained. The results show that the serum CRP level of pregnant mice did not change significantly over time, indicating that intraperitoneal injection of hydrogen peroxide does not cause a significant immune response in pregnant mice.

[0064] Simultaneously, the catalase (CAT) in the offspring embryos of mother mice injected with H2O2 was detected one day later. The detection method was tissue fluorescence staining: two pairs of H2O2-injected pregnant mice (denoted as H2O2) and sterile water-injected control pregnant mice (denoted as CTR) were used. The first pair was labeled Pair #1, and the second pair was labeled Pair #2. Twenty-four hours after injection, both pairs of mice were euthanized, and the offspring embryos were removed. The offspring embryos were fixed overnight with 4% paraformaldehyde solution, then embedded, and the sagittal sections of the embryos were sectioned. The sections were dewaxed with xylene and then hydrated in a gradient of 100%, 75%, 50%, and 20% ethanol. After hydration, the sections were transferred to distilled water for soaking, and then immersed in sodium citrate antigen retrieval solution and microwaved for 10 minutes for antigen retrieval. The sections were removed and immersed in 3% hydrogen peroxide solution for 25 min in a dark environment to inactivate peroxidase. The treated sections were then washed three times with phosphate-buffered saline (PBS), and then completely covered with phosphate-buffered saline (BSA-PBS) containing 5% bovine serum albumin. The sections were then blocked in a humidified chamber at 37°C. Next, fluorescent localization staining of H2O2 reactive protein (CAT) was performed. The blocked sections were incubated overnight at 4°C (in a humidified chamber) using CAT antibody (purchased from Servicebio, catalog number GB11681), followed by a second incubation with fluorescent secondary antibody at room temperature for 1 h. Counterstaining with 4',6-diamidinyl-2-phenylindole (DAPI) was then performed, followed by observation and scanning under a fluorescence microscope. The scanning results are shown below. Figure 2 As shown. In Figure 2 In the diagram, DAPI staining (blue fluorescence) indicates the cell nucleus, CAT staining (red) indicates catalase, Merge is a schematic diagram of the color superposition of the two, H2O2 represents the pregnant mice treated with intraperitoneal injection of hydrogen peroxide, and CTR represents the pregnant mice treated with intraperitoneal injection of sterile water as a control group.

[0065] Depend on Figure 2 It can be seen that, Figure 2 In the middle (A), embryos are derived from the first pair (Pair #1) of H2O2-injected pregnant mice (H2O2) and the uninjected control pregnant mice (CTR). Figure 2 (B) shows another pair (Pair #2) of embryos (H2O2 and CTR). Red fluorescence indicates CAT protein staining, DAPI indicates background staining of the cell nucleus, and Merge indicates overlap of the two signals. The staining of both pairs of embryos showed that the distribution range and staining intensity of the red CAT signal were significantly increased in the H2O2-treated group compared to the control group. This indicates that maternal injection of H2O2 (1 day after injection) resulted in a significant oxidative stress response in offspring mouse embryos, specifically manifested as a significant increase in catalase CAT, the enzyme that decomposes H2O2, within the embryos. Particularly noteworthy is the significantly increased expression of CAT in the embryonic brain region, demonstrating the successful induction of oxidative stress in the embryos.

[0066] In summary, this invention uses only hydrogen peroxide to induce oxidative stress in pregnant female mice, and it has been verified that it does not cause a significant immune response. Furthermore, H2O2 decomposes into H2O2 and O2 after oxidation-reduction reaction, neither of which is toxic or irritating to the body. Therefore, an oxidative stress-specific induced ASD mouse model is formed.

[0067] Example 3: Assessment of the kinetic ability of offspring mice induced by H2O2 during pregnancy

[0068] After the offspring of H2O2-induced offspring reached adulthood (9 weeks of age), their motor abilities were observed and assessed in real time using the open field test and the rotating rod test. Meanwhile, offspring of 9-week-old mice that did not receive H2O2 injection during pregnancy served as the first control group (negative control), and BTBR autistic mice served as the second control group (positive control).

[0069] Open Field Test: After the mice have fully adapted to the behavioral environment, they are placed in the center of a 60cm × 60cm open field, and filming begins. The mice are allowed to move freely within this area, and their movement trajectories are recorded. The timer is stopped after 10 minutes. Before the next test, the open field is cleaned with alcohol and allowed to air dry. The total distance traveled by the mice is analyzed using the Panlab SMART motion trajectory analysis system to assess their motor abilities. The assessment results are as follows: Figure 3 As shown in Figure (A), the vertical axis represents the total distance the mice traveled in the open field, and the horizontal axis, from left to right, represents the offspring of pregnant mice that were not subjected to H2O2-induced oxidative stress (HC) as a negative control, the offspring of pregnant mice subjected to H2O2-induced oxidative stress (OS) as a positive control, and the well-established autism model mouse strain (BTBR). Figure 3 (A) shows that the total distance traveled by the three groups of offspring mice in the open field did not change significantly.

[0070] Rotating Rod Test: One week prior to the test, all mice were given initial training on the rotating rod in the same environment. The training was set as follows: a first-gradient acceleration of 0-5 revolutions per minute for 5 minutes, followed by maintaining a speed of 5 revolutions per minute for 5 minutes, and finally a second-gradient acceleration of 5-40 revolutions per minute for 5 minutes. This allowed the mice to become familiar with the rotating rod environment and the appropriate coping mechanisms. After sufficient adaptation to the behavioral environment, the rotating rod test began. The test procedure was set as follows: the initial speed of the first gradient was set to 5 revolutions per minute for 30 seconds; this was immediately followed by a second-gradient acceleration of 60 revolutions per minute for 250 seconds, until the last mouse fell, ending the single test. Evaluation results are as follows: Figure 3As shown in Figure (B), the vertical axis represents the time taken for the mouse from the start of the experiment to the moment the rotating rod falls, also known as the delayed fall time. The horizontal axis is the same as the vertical axis. Figure 3 (A) is the same. Figure 3 (B) shows that the delayed drop time of OS offspring mice was shorter than that of HC offspring mice, but significantly longer than that of the positive control BTBR autism model mice.

[0071] Depend on Figure 3 It was observed that, compared with the control-treated HC offspring mice and the autism model mice BTBR, the kinetic ability of the H2O2-induced OS offspring mice during pregnancy was not significantly reduced.

[0072] Example 4: Assessment of grooming stereotyped behaviors in offspring mice induced by H2O2 during pregnancy

[0073] In 9-week-old adult mice (progeny induced by H2O2 during gestation), the frequency and duration of grooming behavior were observed in real-time over a 10-minute period to assess the level of stereotyped grooming behaviors, reflecting the core symptom of repetitive behaviors in autism. Adult mice (9 weeks old) of offspring not injected with H2O2 during gestation served as the first control group (negative control), and BTBR autistic mice served as the second control group (positive control). The assessment results are as follows: Figure 4 As shown. Figure 4 In graph (A), the vertical axis represents the total time spent grooming by mice within 10 minutes, and the horizontal axis represents the offspring of negative control mice (HC), offspring of H2O2 oxidative stress pregnant mice (OS), and positive control BTBR autism model mice (BTBR). Figure 4 As shown in (A), the total grooming time of the OS group mice was significantly longer than that of the HC control group. Figure 4 In diagram (B), the vertical axis represents the frequency of grooming behavior in mice within 10 minutes, derived from... Figure 4 As shown in Figure (B), the grooming frequency of OS mice was significantly higher than that of HC offspring mice.

[0074] Depend on Figure 4 It was observed that, compared with the offspring mice treated with negative control, the offspring mice induced by H2O2 during pregnancy exhibited significantly increased stereotyped and repetitive behaviors, reflecting the typical core symptoms of ASD.

[0075] Example 5: Assessment of three-box social abilities in offspring mice induced by H2O2 during pregnancy

[0076] After the offspring of H2O2-induced offspring reached adulthood (9 weeks old), the Three Chamber Social experiment was conducted. Under conditions of adequate acclimatization to the behavioral environment, a stranger mouse (of the same age and sex as the H2O2-induced offspring, without any prior induction treatment) and a toy were placed in the left and right interaction boxes, respectively. The test mouse was placed in the middle box, allowing for free interaction. The timer was 10 minutes, marking Phase I. Panlab SMART trajectory analysis software was used to analyze the test mouse's social preferences. After the test mouse rested briefly and cleaned the test box, the toy was replaced with another stranger mouse (of the same age and sex as the H2O2-induced offspring, without any prior induction treatment), and the timer was again 10 minutes, marking Phase II. The test mouse's social preference for the new stranger mouse, i.e., social novelty, was analyzed. The results from both phases were used to comprehensively assess social ability. Simultaneously, adult mice (9 weeks old) that did not receive H2O2 injection during pregnancy served as the first control group (negative control), and BTBR autistic mice served as the second control group (positive control). The evaluation results are as follows: Figure 5 As shown, Figure 5 The diagram in Figure (A) is a schematic diagram of the three-box social experiment. The left box contains unfamiliar mice 1 in both phase I and phase II, and the right box contains toys and unfamiliar mice 2 in phase I and phase II, respectively. The tendency of mice to socialize on the left side in phase I is denoted as Preference (P), and the tendency of mice to socialize on the right side in phase II is denoted as Novelty (N). Figure 5 The vertical axis of (B) represents the percentage of the mouse's social tendency, calculated as (left-side social time - right-side social time) / total social time × 100%. Figure 5 (B) shows that HC offspring mice from pregnant mice that did not receive H2O2 injection during pregnancy had complete social function, with significant differences in P and N; while OS offspring mice from pregnant mice that received H2O2 injection during pregnancy showed obvious social function deficits, as did the BTBR model mice used as a positive control.

[0077] Depend on Figure 5 It was observed that, compared with the offspring mice treated with negative control, the offspring mice induced by H2O2 during pregnancy had significantly weakened social abilities, reflecting the typical core symptoms of ASD.

[0078] Example 6: Histological examination of H2O2-induced synaptic tissues in offspring mice during pregnancy

[0079] After adulthood (9 weeks old), offspring mice induced by H2O2 during pregnancy were euthanized, and their brain tissue was rapidly isolated. The brain tissue was then fixed and stained with Golgi apparatus, and synaptic tissue was observed using an optical microscope. Offspring mice (9 weeks old) that did not receive H2O2 injection during pregnancy served as the first control group (negative control), and BTBR autistic mice served as the second control group (positive control). Evaluation results are as follows: Figure 6 As shown, Figure 6 (A) is a schematic diagram of the morphology of neuronal dendrites radiating from the cell body under microscopic observation of Golgi staining in the brain tissue of mice in each group. Magnify is a magnified schematic diagram of dendrites in a specific segment. Figure 6 As shown in (A), compared with the negative control HC offspring mice, the density of neuronal dendritic spines in H2O2-induced OS offspring mice and BTBR autism model mice during pregnancy was significantly reduced. Figure 6 The vertical axis of (B) represents the statistical count of dendritic spines within a 50μm distance of dendrites in the microscopic staining image. Figure 6 Similarly, in (B), the number of neuronal dendritic spines was significantly reduced in OS offspring mice and BTBR autism model mice compared with negative control HC offspring mice.

[0080] Depend on Figure 6 It can be seen that, compared with the control offspring mice, the dendritic spines in the brain tissue of H2O2-induced offspring mice during pregnancy were significantly reduced under an optical microscope, which is consistent with the typical core pathological manifestations of ASD.

[0081] Example 7: Detection of H2O2-induced synaptic markers in offspring mice during pregnancy

[0082] Adult mice (9 weeks old) of H2O2-induced OS offspring and HC offspring (9 weeks old) of the negative control group were anesthetized with afodin. Brain tissue was then fixed to the heart using 4% paraformaldehyde solution. After further fixation overnight, the brain tissue was stained with immunofluorescence for synaptic markers Synaptophysin (red fluorescence) and PSD95 (green fluorescence), and the cell nuclei were counterstained with DAPI (blue fluorescence). The tissues were then observed under a scanning electron microscope. Adult mice (9 weeks old) of offspring not conceived with H2O2 injection during pregnancy served as the control group. The evaluation results are as follows: Figure 7 As shown.

[0083] Figure 7 In the brain, Hippocampis refers to the hippocampus, and Amygdala refers to the amygdala. Figure 7It was found that, compared with the control offspring mice, the distribution of synaptic markers Synaptophysin and PSD95 staining in H2O2-induced offspring mice during pregnancy was significantly altered under scanning electron microscopy, indicating changes in synaptic connections and functions, consistent with the typical core molecular pathological changes of ASD.

[0084] Example 8: Detection of molecular pathological changes in the brains of offspring mice induced by H2O2 during pregnancy

[0085] After reaching adulthood (9 weeks old), offspring mice induced by H2O2 during pregnancy were euthanized, and their brain tissue was rapidly isolated for RNA extraction, reverse transcription, and transcriptome sequencing. Offspring mice that did not receive H2O2 injection during pregnancy served as a control group. The differentially expressed genes in the brain tissue transcriptomes of the two groups were analyzed and compared. The results are as follows: Figure 8 As shown, Figure 8 The graph in (A) is a volcano plot showing differentially expressed genes between the two groups. The vertical axis indicates the significance of the expression difference (p-value, logarithmic form), and the horizontal axis indicates the fold change in expression (logarithmic form). Figure 8 As shown in (A), there are significant differences in gene expression between the two groups. Figure 8 (B) is Figure 8 Among the differentially expressed genes in (A), downregulated genes (green) and upregulated genes (red) with a clear association with the onset of autism were found; Figure 8 It can be seen that the differentially expressed upregulated and downregulated genes include several currently known key pathogenic genes of ASD, suggesting at the molecular level the comprehensive driving role of oxidative stress in the occurrence of ASD.

[0086] GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were performed on differentially expressed genes, and the results are as follows: Figure 9 and Figure 10 As shown.

[0087] Figure 9 This diagram illustrates the network of biological processes enriched by differentially expressed genes. Circles represent specific enriched biological processes, the size of the circle represents the number of genes contributing to that enrichment, the color intensity represents the significance of that enrichment, and the lines between the circles represent the inherent biological connections between these nodes. Figure 9It is known that differentially expressed genes are concentrated in molecular pathways related to synaptic transmission, including biological processes related to synaptic signaling and neurotransmitter transmission such as synaptic transmission, dopaminergic, trans-synaptic signaling, chemical synaptic transmission, synaptic signaling, amine transport, dopamine transport, and catecholamine transport, which is consistent with the molecular pathogenesis of ASD.

[0088] Figure 10 The KEGG pathways enriched by differentially expressed genes are shown on the left. The entries on the left are the names of the KEGG pathways, the horizontal axis represents the enrichment degree of the corresponding pathway, the size of the circle represents the number of genes contributing to the enrichment of that pathway, and the gradient color represents the significance of the enriched pathway, with red being the most significant and blue the least significant. Figure 10 The enrichment results are mainly observed in neuroactive ligand-receptor interactions, dopaminergic synapses, retrograde endocannabinoid signaling, and oxidative phosphorylation, consistent with the molecular pathogenesis of ASD. In summary, differentially expressed genes are concentrated in synaptic connections and signal transduction-related functions and pathways.

[0089] In summary, the ASD progeny mice induced by hydrogen peroxide-induced simple oxidative stress in this invention exhibit the following characteristics: 1) pronounced stereotyped grooming behavior; 2) significantly reduced social tendency; 3) significantly abnormal synaptic distribution in brain tissue; 4) significantly differential expression of ASD-related genes in brain tissue; and 5) significantly differential regulation of synaptic pathways in brain tissue. This indicates that the modeling method provided by this invention is relatively simple and can obtain ASD model mice under simple oxidative stress.

[0090] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for establishing a hydrogen peroxide-induced mouse autism model, characterized in that, Includes the following steps: The method for establishing the hydrogen peroxide-induced mouse autism model includes: The hydrogen peroxide solution was injected into the peritoneal cavity of pregnant mice on day 12.5 of embryonic development to induce hydrogen peroxide, and offspring mice after hydrogen peroxide induction were obtained, which are mouse autism models. The amount of hydrogen peroxide injected into the peritoneum of the pregnant mice was 180~220 μmol / kg; The hydrogen peroxide induction time is 6-72 hours.

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