{"paper_id":"0e64a0fc-bcd0-4c86-a1e3-55ecc27847dc","body_text":"Triple-Phase VPA Administration in C57 Mice: A Precision Timing Strategy for Cost-Effective ASD Modeling with Reduced Maternal Toxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Triple-Phase VPA Administration in C57 Mice: A Precision Timing Strategy for Cost-Effective ASD Modeling with Reduced Maternal Toxicity Zhaoming Liu, Zuoxian Lin, Lin Fu, Heying Li, Yujie Liu, Yirong Sun, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6545657/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Valproic acid (VPA)-induced rodent models are widely utilized to study autism spectrum disorder (ASD) but suffer from high maternal mortality (> 25%) and inconsistent phenotypic outcomes due to imprecise dosing timing and acute embryotoxicity. To address these limitations, we developed a novel triple-phase VPA administration protocol (300→400→300 mg/kg) in C57 mice, strategically aligned with critical neurodevelopmental windows (E11.5–E13.5). This study aimed to optimize ASD modeling by balancing cost-effectiveness with biological fidelity while minimizing maternal toxicity. Behavioral assessments (open field, three-chamber social test, Morris water maze), ultrastructural analysis (TEM), and neuroinflammatory/oxidative stress profiling were conducted to validate model robustness. The results demonstrated that the triple-phase regimen achieved 100% maternal survival (vs. 75% in traditional single-dose protocols, P < 0.05) and significantly reduced abortion rates (P < 0.01). The offspring exhibited ASD core phenotypes, including social deficits (reduction in stranger interaction time, P < 0.0001), repetitive behaviors (increased marble burying, P < 0.01), and spatial memory impairments (prolonged escape latency P < 0.01). Crucially, synaptic ultrastructural pathologies—presynaptic vesicle depletion and mitochondrial crista disorganization—were observed alongside dysregulated prefrontal IL-6/TNF-α levels (P < 0.01) and oxidative stress markers (P < 0.01). The protocol reduced costs through optimized dosing while maintaining phenotypic consistency. This work establishes a standardized, ethically compliant ASD model that reconciles economic efficiency with neurodevelopmental validity, offering a transformative tool for mechanistic studies and therapeutic screening. Biological sciences/Neuroscience Biological sciences/Zoology Health sciences/Neurology ASD model Valproic acid (VPA) Phase-specific dosing strategy Maternal toxicity reduction C57BL/6 mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Highlights This article presents the first application of chronopharmacological principles to ASD mouse models via phase-specific VPA dosing. 100% maternal survival with significant cost reduction, addressing Nature Protocols' call for affordable models. Phenotypic consistency has improved, addressing reproducibility challenges in existing models. Behavioral, ultrastructural, and molecular profiling can be combined to confirm the ASD-like pathophysiology. Maternal toxicity and embryotoxicity were mitigated in accordance with the \"3R\" principles (reduction, refinement, replacement). Introduction Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by persistent deficits in social communication and interaction alongside restricted and repetitive behaviors, affecting approximately 1–2% of the global population [1, 2]. Despite decades of research, the etiology of ASD remains elusive, with heterogeneous genetic and environmental contributions complicating the mechanistic understanding [3]. Animal models, particularly rodents, have become indispensable for studying ASD pathophysiology, yet existing paradigms face critical limitations in terms of biological validity, reproducibility, and ethical feasibility [4]. This study introduces a novel triple-phase valproic acid (VPA) administration protocol in C57BL/6J mice that is designed to overcome these challenges through precision timing, reduce maternal toxicity, and increase cost-effectiveness. ASD encompasses a wide range of conditions with significant heterogeneity in symptoms and severity [5]. The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) defines ASD on the basis of deficits in social communication and restricted, repetitive behaviors [6]. Children with ASD often face challenges in social interactions, such as difficulties in making eye contact, understanding social cues, and engaging in reciprocal conversations [7]. They may also exhibit repetitive behaviors such as hand-flapping, rocking, or insistence on sameness [8]. These symptoms can significantly impact their daily lives, education, and future social and occupational functioning [9]. Animal models, especially rodent models, are invaluable tools for studying the pathophysiology of ASD and evaluating potential therapeutic interventions [10]. Mice and rats are commonly used because of their genetic similarity to humans, short reproductive cycles, and ease of genetic manipulation [11]. There are various types of rodent models for ASD, including genetic models and neurotoxicological models [12]. Genetic models, such as Shank3 knockout mice and BTBR T+tf/J mice, mimic specific genetic mutations associated with ASD in humans and display behavioral deficits similar to those observed in ASD patients [13]. Neurotoxicological models, such as the VPA-induced model, are generated by exposing pregnant rodents to VPA, leading to offspring with ASD-like behaviors [14]. Limitations of Current Models (1) Temporal and Dose Inaccuracy Critical neurodevelopmental windows (e.g., neural tube closure at E11.5–E13.5) require precise VPA timing, but individual variability in conception complicates single-dose synchronization. Melancia et al. (2018) demonstrated that only 33% of litters receive VPA during optimal windows, leading to interlitter variability (CV > 30%) in cortical migration deficits [15]. (2) Maternal Toxicity and Ethical Concerns High-dose VPA triggers placental insufficiency and metabolic acidosis, resulting in abortion rates exceeding 50% [14]. Such outcomes contradict the \"3R\" principles (replacement, reduction, refinement), limiting ethical approval and scalability [16]. (3) Cost-effectiveness and generalizability The cost of establishing and maintaining rodent models for ASD research can be high, especially for genetic models that require complex genetic engineering techniques [17]. This may limit the widespread use of these models, particularly in resource-limited laboratories. Additionally, the generalizability of the findings from rodent models to humans is still a matter of debate. Therefore, it is essential to develop more cost-effective and translatable rodent models to improve the relevance of the research findings. The core objective of this study was to develop an optimized, cost-effective C57 mouse model of ASD that can accurately recapitulate the behavioral and pathophysiological features of the disorder. On the basis of previous research, we hypothesize that a triple-phase VPA regimen (300→400→300 mg/kg at E11.5, E12.5, and E13.5) will increase maternal survival: distributing doses across gestational stages reduces peak plasma concentrations, mitigating acute toxicity. Optimize Neurodevelopmental Targeting: Ensure that all litters receive VPA during E11.5–E13.5, irrespective of conception timing. Maintaining Phenotypic Fidelity: Preserving ASD-like behaviors and synaptic pathologies while lowering cumulative VPA exposure. To address these challenges, we developed a triple-phase VPA administration protocol (300→400→300 mg/kg at E11.5, E12.5, and E13.5) that integrates chronopharmacological principles with neurodevelopmental staging. Our protocol introduces three transformative elements: Chronopharmacological alignment: The phased regimen ensures that all offspring receive VPA during E11.5–E13.5, regardless of conception timing—a critical advancement over single-dose methods. Toxicity mitigation: By avoiding suprathreshold VPA concentrations (IC50 for embryotoxicity: 450 mg/kg), we reduce mitochondrial dysfunction in placental trophoblasts, a key driver of pregnancy loss. Multidimensional Validation: Combining automated behavioral tracking (EthoVision XT), synaptic ultrastructural analysis (TEM), and neuroinflammatory profiling to establish causal links between molecular perturbations and ASD-like behaviors. Prior studies have laid essential groundwork but lack translational cohesion. For example, Nicolini and Fahnestock established the role of VPA in altering the GABA/glutamate balance[18], whereas Roullet et al. linked prenatal VPA exposure to prefrontal‒amygdala circuit hyperconnectivity[19]. However, neither the inherent toxicity nor the reproducibility of the protocol has been addressed. Our work directly builds on these findings by introducing a timing-optimized model that preserves synaptic pathologies (e.g., vesicle depletion, PSD thickening) while eliminating confounding maternal mortality. Furthermore, we extended the mechanistic scope by demonstrating dose-dependent neuroinflammatory dysregulation, a feature that is absent in genetic models such as Shank3 knockouts[20]. Materials and Methods 1.1 Experimental Materials 1.1.1 Experimental Animals Adult C57BL/6 mice (96 females, 48 males, body weights of 20–30 g) were purchased from Zhuhai Baite Tong Biotechnology Co., Ltd. (Production License Number: SCKK (Yue) 2020–0051; C57BL/6 mouse certificate number: 44822700030567). All animals were housed in the SPF-level animal facility of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Housing License Number: SYXK (Yue) 2022–0063), under controlled environmental conditions: temperature 22°C ± 2°C, relative humidity 50%–60%, 12 h/12 h light–dark cycle, with ad libitum access to standard rodent diet and sterilized drinking water. This study is reported in accordance with the ARRIVE guidelines (Percie du Sert et al., 2020). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Ethics Approval No. CAS (IACUC:2023081)), and strictly adhered to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 8th edition, 2011). Euthanasia and anesthesia protocols followed the 2020 American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, and no prohibited methods (e.g., chloral hydrate, ether, or chloroform) were employed. Every effort was made to minimize animal suffering, including the use of appropriate analgesic regimens and humane endpoints during the study. 1.1.2 Reagents and Equipment Valproic acid (VPA, Sigma‒Aldrich Company, USA) was used. The behavioral testing system included a plane righting reflex test platform (20×20×2 cm), an auditory startle reflex device (1 cm³ stainless steel block placed on a 35×30×0.5 cm metal plate), three chamber test boxes (60×40×22 cm), and an open field test box (50×50×30 cm). 1.2 Preparation of the ASD animal model induced by VPA Adult male and female C57BL/6 mice were placed in the same cage at 17:00 p.m. in the SPF animal room of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. On the 2nd day after cage placement, vaginal checks were conducted at 9:00 a.m. The presence of vaginal plugs was considered successful fertilization, and the date of vaginal plug appearance was recorded as the 0.5th day of pregnancy. 1.2.1 C57BL/6 Mice were randomly divided into 4 groups according to the method of VPA administration : (1) Traditional Model Group: E12.5 injection of VPA (valproic acid sodium) (600 mg/kg) (2) Modified Model Group 1: Multiple administrations (E12: 200; E12.5: 300; E13: 200 mg/kg) (3) Modified Model Group 2: Multiple administrations (E11.5: 300; E12.5: 400; E13.5: 300 mg/kg) (4) Control group: E12.5 injection of an equal volume of normal saline 1.2.3 Observation indices after group administration ① Observe the adverse reactions, mortality, and abortion rates of pregnant mothers in each group after the administration of valproic acid sodium or normal saline; ② Observe the morphology, growth and development of newborn pups in each group; ③ The social behavior of newborn pups in each group was observed. 1.3 Reproductive outcome analysis in each group ① Delivery rate assessment: The number of pregnant C57 mice that were successfully delivered was recorded for each experimental group. The delivery rate was calculated as follows: Delivery rate (%) = number of delivered mice/number of pregnant mice × 100%. ② Neonatal morphological abnormality evaluation: The appearance of the skull and limbs of pups (including male and female pups) in each group was observed on the day of birth, and the presence or absence of appearance deformities (i.e., short development of limbs, inconspicuous eyeballs and auricles) of pups in each group (except for tail deformities) was determined. The malformation rate was determined as follows: Malformation rate (%) = (number of malformed pups/total live births) ×100%. 1.4 Adverse reactions and mortality in pregnant mice following drug administration After intraperitoneal (i.p.) injection, pregnant mice were returned to their home cages. Adverse reactions were monitored for 30 min postinjection, and mortality was recorded within 6 h. The results are summarized in the following table. 1.5 Abortion rate analysis in pregnant mice after treatment Abortion was determined on the basis of the following criteria: ① Vaginal plug presence was confirmed at 9:00 AM the morning after mating. ② Significant body weight gain and abdominal distension were detected by gestational day (GD) 12.5. ③ Sudden body weight loss between GDs 19–20. Pregnant mice fulfilling all three criteria were classified as aborted. The abortion rate (%) was calculated as the number of aborted females/number of pregnant females in each group×100%. 1.6 Effects of VPA Exposure on the Physiological Development of Male Offspring (1) Four-day survival rate of male pups: 4-day survival rate (%) = (Male pups surviving at postnatal day 4/Live male births) ×100% (2) Body mass: Body mass was measured via an electronic balance (each animal was measured three times, and the average was calculated). (3) Tail length: The length was measured from the tail base to the tip in the extended position (each animal was measured three times, and the average length was calculated). (4) Tail curvature evaluation (PND 30): Tail curvature rate (%) = (Adult males with tail curvature/Total adult males) ×100% (5) Incisor eruption: Positive when lower incisors exhibited visible white spots and tactile resistance (6) Eye opening: Positive when ≥50% of the orbital area is exposed (7) Fur development: Positive when abdominal skin pigmentation changes from pink to black (C57 mice) with a fur coat. 1.7 Effects of VPA Exposure on Neurological Development in Male Offspring The neurological development of male pups was evaluated via four standardized reflex tests: surface righting reflex, cliff avoidance reflex, air righting reflex, and pivoting behavior. The postnatal age at which each reflex reached positive criteria was recorded for all pups in each group. (1) Surface Righting Reflex Procedure: Pups were placed in a supine position on a flat surface for 3 sec and released. Positive criterion: Complete rotation to the prone position with all four paws contacting the surface within 10 sec. (2) Cliff avoidance Reflex Procedure: Pups were positioned at the edge of a 30 cm-high platform with their heads protruding. Positive criterion: Turning or retreating movement within 10 sec. (3) Air righting reflex Procedure: Pups were held supine 30 cm above a soft cushion for 3 sec before release. Positive criterion: Successful landing in the prone position in 3 consecutive trials. (4) Negative Geotaxis Reflex Procedure: Pups were placed head-down on a 25° inclined plane (PND 7--10). Positive criterion: Completion of 180° turning to the head-up position within 30 sec. Functional significance: This test evaluates vestibular function and motor coordination. A prolonged turning time may indicate impaired vestibular development. Note: All reflex tests were performed daily until all pups in each group met the positive criterion. The developmental age for each reflex was recorded when 100% Pups in a group achieved a positive response. (5) Bar Holding Test Procedure: Pups were suspended by their forelimbs on a horizontal bar (25 cm height). Positive criterion: Maintaining grip for ≥2 seconds Significance: Evaluates forelimb strength and grip endurance (6) Crawling behavior Procedure: Pups were placed on a flat surface Positive criterion: Independent forward locomotion (≥3 cm) using all four limbs while maintaining ventral contact with the surface Significance: Assessment of early locomotor coordination The postnatal age at which 100% of the pups in each group achieved positive responses was recorded for both tests. (7) Auditory startle reflex assessment The auditory startle reflex was evaluated daily from postnatal day (PND) 2 using a standardized protocol: Test Procedure: A metal plate was positioned horizontally 15 cm below the test subject. A metal block was dropped vertically onto the plate to generate an abrupt acoustic stimulus (90–100 dB). Testing was conducted in a sound-attenuated chamber. Positive criterion : Immediate whole-body startle response (distinct curling or trembling) following sound presentation. The developmental milestone was recorded as the postnatal age when 100% of the male pups in each group exhibited consistent positive responses. (8) Tail–Flick Analgesia Test The tail-flick test assesses nociceptive responses in rodents by measuring withdrawal latency to radiant heat stimulation. This method offers distinct advantages over hot plate testing, particularly its applicability to lightly anesthetized animals and independence from motor coordination. Testing Procedure: ① The experimental animals were removed from the animal room, weighed, allowed to acclimatize in the laboratory for 30 min, and the control group was separated from the model group. ② Determine the baseline latencies of the animals with a tail flash tester (i.e., there is a heat source under the plate of a small hole), put the tail of the mouse (approximately 50 mm in front of the tail tip) or mouse (approximately 15 mm in front of the tail tip) on top of the small hole, start the heat source to start timing until the tail dodges, adjust the intensity of the light source, set most of the time of the tail dodge time to 3–4 s; if there is no dodge reflex, then set the test termination time to 10 s to avoid burns; ③ Test the tail flash reaction, put the animal on the tail flash test board and put its tail on the small hole of the light source, that is, start timing, observe the time of the animal's tail dodging reaction, or until the termination (cutoff) time. Note that only 1 time point is measured in a test, not 3. 1.8 Behavioral Assessments A researcher, blinded to the group assignments, conducted the behavioral evaluations. ASD-like behavior was scored via Noldus EthoVision XT software (Noldus, Netherlands). The behavioral assessments were carried out during the day between 09:00 and 18:00. 1.8.1 Open field test The open field test was used to evaluate exploratory behavior and anxiety-like responses in rodents exposed to a novel environment. This paradigm exploits the natural conflict between rodents' exploratory drive and their aversion to open spaces. An overhead camera system was used to record the path, and Noldus EthoVision XT software was employed to determine the frequency of entries and the time duration within the central area. The open field test (OFT) is a common assay for evaluating both anxiety-like and motor behaviors in animals (Kraeuter et al., 2019). Protocol: a) Habituation (10 min): Animals freely explored the apparatus b) Testing (10 min): Recorded parameters included the following: Central/peripheral crossings (all limbs crossing sector borders) Vertical activity (forelimbs raised ≥2 s) Analysis: Central crossings: Anxiety index (fewer crossings = greater anxiety); Vertical activity: Exploratory behavior indicator; Peripheral activity: General locomotor function. Environmental controls: The samples were thoroughly cleaned with 75% ethanol between trials under consistent testing conditions (lighting, noise, etc.), and the experimenter remained outside the visual field. 1.8.2 Three- chamber social interaction test The three-chamber test was used to evaluate social preference and novelty recognition in rodents through a standardized two-phase protocol. The experiment was carried out in a connected transparent three-chamber chamber, which is a classic way of assessing social skills in rats (Buffington et al., 2016). Experimental Protocol: Habituation: 7 days pretest: Daily 2-h acclimation to the test room. Days 1--3: 3-min center chamber exposure followed by 3-min full chamber access. Test day: 5-min free exploration with empty wire cages in the side chambers Testing Phases: Socialization test: On one side of the chamber, a male Stranger 1 of the same age and from a different litter (of the same sex and age as the test animal and of the same strain that has not yet been housed in the same cage) was placed in an inverted metal coil, which was labeled the Stranger 1 cage. Only one inverted transparent metal coil was placed on the other side of the chamber, which was labeled the object cage. The test mice were placed in the empty central cage, and the activity of the test mice and the duration of mutual olfactory communication with stranger 1 or the object were observed for 10 min. ② Social preference test: After the first stage of the socialization test, another Stranger 2 of the same age, same sex and different litters was placed in an empty inverted metal coil and was observed for 10 min. The time spent in each side chamber and the time spent sniffing and communicating with Stranger 1 and Stranger 2 were recorded by a camera system and computer software. At the end of the experiment, the test mice and Stranger 1 and Stranger 2 inside the inverted metal coils were removed sequentially and returned to different cages. After each round of experiments, feces, urine and other excreta were promptly removed, and the experimental apparatus was wiped with 75% alcohol and dried to remove as much odor as possible from the previous animal before replacing it with the next mouse for behavioral testing. 1.8.3 Stereotyped Behavior Assessment Two standardized tests were employed to evaluate the repetitive behavior characteristics of the ASD models: Self- grooming test Protocol: a) 10-min habituation period b) 10-min observation period Measured parameters : Cumulative time spent grooming face, limbs, body, and tail (2) Marble Burying Test The tested mice were placed in a box. The bottom of the box was lined with a layer of clean bedding approximately 5 cm thick, and the mice were placed in the box with bedding for 3 minutes to acclimatize before starting the experiment, after which the mice were removed and placed in a transit cage to wait. The bedding in the box was flattened, and 16 black glass balls 1.6 mm in diameter were placed in a 4 × 4 grid. The mice were then placed in the box and allowed to roam freely for 10 min. After 10 min, the mice were removed and photographed from all angles with a camera, and the number of beads buried was counted from the pictures and videos by three statisticians who were trained to count the number of beads from the pictures and videos and who had no knowledge of the subject: glass beads with more than 75% of the beads buried in bedding were considered buried. The enumerators had no knowledge of the grouping, and the final results were averaged and rounded. 1.8.4 Morris Water Maze Behavioral Assessment Spatial learning and memory were evaluated via the Morris water maze paradigm following established protocols from prior investigations. The experimental apparatus consisted of a round pool filled with temperature-regulated water (23–25°C) rendered opaque through the addition of a food-grade titanium dioxide suspension. A circular escape platform (14 cm diameter) remained consistently positioned 1.5 cm below the water surface in the target quadrant throughout the acquisition training. The training protocol comprised four daily sessions over five consecutive days, with each session containing four randomized entry-point trials. The subjects were allotted 60 s per trial to locate the submerged platform, followed by a 10 s consolidation period upon successful navigation. Animals that failed to locate the platform within the allotted time were manually assisted in reaching the platform via tail guidance. An intertrial interval of 25 min was maintained in heated recovery cages between successive trials. Spatial memory retention was assessed 24 h posttraining through a 60 s free-swim probe trial in which the platform was removed. All behavioral sessions were digitally captured via a video tracking system (EthoVision XT v15.0, Noldus Information Technology) for subsequent quantitative analysis. The primary outcome measures included the following: Acquisition phase: Escape latency (s) across training days. Probe trial: Platform location traversals; first-cross latency (s); swimming velocity (cm/s). 1.9 Biochemical analysis The GSH, T-SOD, GSH-PX, MDA, CAT, T-NOS, and NO levels were measured via the corresponding biochemical kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China). The levels of IL-6, IL-10, IL-1β and TNF-α were also measured via mouse ELISA kits (Fankel, Shanghai, China). 1.10 Ultrastructural analysis via transmission electron microscopy (TEM) At postnatal day 60 (PND 60), the mice were anesthetized via intraperitoneal injection of a ketamine‒xylazine mixture (75 mg/kg or 5 mg/kg) and subjected to transcardiac perfusion through the ascending aorta. The perfusion protocol consisted of an initial flush with ice-cold 0.01 M sodium‒potassium phosphate buffer (pH 7.4, containing 0.9% NaCl), followed by fixation with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, 20°C; Sigma‒Aldrich, St. Louis, MO, USA). Tissue blocks (approximately 1 mm³) were dissected from the frontal cerebral cortex and hippocampal CA1 region across all experimental and control groups for ultrastructural examination. Following 20 hours of primary fixation in ice-cold fixative, the samples were postfixed in a solution containing 1% osmium tetroxide (OsO₄) and 0.8% potassium ferrocyanide [K₄Fe(CN)₆]. After dehydration through a graded ethanol series, the tissue blocks were embedded in epoxy resin (Epon 812). Ultrathin sections (60 nm thickness) were prepared via an ultramicrotome, double-stained with uranyl acetate and lead citrate, and examined via transmission electron microscopy (JEM-1200EX, Jeol, Japan). Digital images were acquired via a MORADA CCD camera coupled with iTEM 1233 imaging software (Olympus, Japan). The data are presented as the means ± SEMs from 3 independent animals per group (VPA-exposed vs. control). 1.11 Statistical analysis Quantitative data expression: All continuous variables are expressed as the mean ± standard deviation (x̅±s). 1. Normality and Homogeneity of Variance Assessment Data normality was evaluated via the Shapiro‒Wilk test prior to statistical analysis. 2. Comparative Analysis 2.1 Two-Group Comparisons Parametric conditions: When the data followed a normal distribution and homogeneity of variance was confirmed by Levene's test (P>0.05), an independent two-sample t test was applied. Nonparametric conditions: For nonnormally distributed data or unequal variances, the Mann‒Whitney U test (Kruskal‒Wallis) was employed. 2.2 Categorical Variable Comparisons Pearson’s χ² test was used when all expected frequencies were ≥ 5. Fisher’s exact test was adopted if any expected frequency was < 5. 2.3 Multigroup Comparisons Parametric data: One-way analysis of variance (ANOVA) with Tukey’s HSD post hoc test was performed. Nonparametric data: The Kruskal‒Wallis test followed by Dunn’s multiple comparison correction was used. Results 2.1 Dose- and time-dependent maternal toxicity of VPA Prenatal sodium valproate (VPA) administration has dose- and gestational timing-dependent effects on maternal survival and pregnancy maintenance. (1) Toxic reaction of pregnant mice (Table 1): After a single intraperitoneal injection of VPA (600 mg/kg) in the traditional model group, pregnant C57BL/6 mice presented acute toxicity symptoms at 3–5 min, which included stiffness of the limbs, motor disorders, paralysis with the eyes closed, and respiratory and circulatory disorders, which ultimately led to death. In contrast, the symptoms of the fractionally administered groups were significantly relieved: the time to the onset of symptoms of rigidity was significantly delayed in modified group 1 (13–15 min) and modified group 2 (8–10 min), and they maintained basic motor ability during the 30-min observation period. (2) Survival analysis (Table 1): Within 6 h after drug administration, the mortality rate of the traditional group reached 25% (6/24), whereas there were no deaths in modified groups 1 and 2 (0/24), and the difference was statistically significant (p<0.05) (Fisher's exact test). (3) Pregnancy outcome (Table 1): the abortion rate was 54.17% (13/24) in the traditional group and significantly reduced to 29.17% (7/24; p<0.05) (Fisher's exact test) in modified group 1. Notably, the miscarriage rate in modified group 2 further decreased to 12.5% (3/24), which was highly significantly different from that in the traditional group (Fisher's exact test, p<0.01). Table 1. Comparison of production in pregnant C57BL/6 mice Group Abortion (n, %) Mortality(n, %) Normal (n, %) Control 0 (0%) 0 (0%) 24 (100%) Traditional 13（54.17%） 6 (25%) 5 (20.83%) Modified 1 7 (29.17%)* 0 (0%)* 17 (70.83%) Modified 2 3 (12.5%)** 0 (0%)* 21(87.5%) 2.2 Dose- and time-dependent teratogenic effects of prenatal VPA exposure All newborn mice in the control group presented normal developmental phenotypes, including reddish skin color, complete body shape and no malformations visible to the naked eye (n=106). In contrast, the offspring of the traditional model group presented significant developmental abnormalities: 15.63% (5/32) of the littermates presented typical deformities, such as shortened limbs, underdeveloped eyes (including one case of monocular deformity), and missing auricles. Statistical analysis revealed that the malformation rate of this group was significantly greater than that of the control group, confirming that a single high-dose intraperitoneal injection of VPA (600 mg/kg) induced severe embryotoxicity (P<0.05) (Fisher's exact test) (Table 2). Notably, the teratogenic effect due to embryotoxicity was significantly reduced in the modified dosing regimens (Group 1 and Group 2), and the offspring（Figure1A）malformation rate was 0% in both groups, which was not significantly different from that of the control group (P >0.05). The C57BL/6 mouse model of ASD, generated through an optimized experimental protocol, exhibited a classical tail curvature phenotype（Figure1C）in the majority of subjects. This characteristic morphological feature served as a primary phenotypic marker for successful model validation. 2.3 Dose- and Time-Dependent Developmental Impairments in Male Littermates by VPA Exposure (1) 4-day survival rate (Table 2): The 4-day survival rate of C57BL/6 male littermates in the traditional model group was 78.13% (25/32), which was significantly lower than that in modified group 1 (100%, 76/76) (P<0.001) (Fisher's exact test) and modified group 2 (100%, 87/87) (P<0.001) (Fisher's exact test), suggesting that the optimization of the VPA exposure regimen could effectively reduce the early mortality of neonatal mice. (2) Inhibition of body weight development (Figure 2A): male littermates in all intervention groups (traditional, modified group 1 and modified group 2) presented significant body weight inhibition beginning 21 days after birth (P<0.01). (3) Lagging tail length development (Figure 2B): Tail lengths (Figure 1B) of male littermates across experimental groups were measured. the tail lengths of male littermates in all intervention groups (traditional, modified group 1 and modified group 2) were significantly shorter than those of the control group beginning at 28 d after birth (P<0.01). (4) Tail curvature rate (see Table 2): The incidence of bent tails in the traditional group was 40% (10/25), which was significantly lower than that in modified group 1 (82.9%, 63/76) (P<0.001) (Fisher's exact test) and modified group 2 (88.51%, 77/87) (P<0.001) (Fisher's exact test). Curved tail deformities in rodents are thought to be associated with minor neural tube closure defects. (5) Age of positive incisor eruption (Figure 2C): The age of positive incisor eruption in C57BL/6 pregnant mice in modified group 1 and modified group 2 was significantly delayed compared with that in the control group (P<0.01), but there was no significant difference between the traditional model group and the control group (P>0.05). (6) Age of positive fur development (Figure 2D): Male C57BL/6 model mice in the traditional model group and modified group 1 exhibited positive abdominal hair growth, which was significantly delayed compared with that of the control group (P<0.05). Compared with the control group, modified group 2 was significantly delayed (P<0.01). These findings suggest that the injection of VPA during pregnancy can significantly delay the development of incisor eruption, eye opening and fur development in male C57BL/6 model mice in the modified model group. (7) Age of positive eye opening (Figure 2E): Compared with that in the control group, the age of positive eye opening in the modified group 2 was significantly delayed (P<0.01), the age of positive eye opening in the modified group 1 was significantly delayed (P<0.01), and there was no significant difference between the traditional model group and the control group (P>0.05). Table 2 4-day survival rate , tail curvature rate and incidence of deformities in newborns (n,%) Group 4-day survival rate (n, %) Tail curvature rate (n, %) appearance deformities rate (n, %) Totality Control 106 (100%) 0 (0%) 0 (0%) 106 Traditional 25（78.13%） 10 (40%) 5 (15.63%)* 32 Modified 1 76(100%)*** 63(82.90%)**** 0 (0%) 76 Modified 2 87(100%)*** 77 (88.51%)**** 0 (0%) 87 2.4 Dose- and time-dependent effects of VPA on neurological development The experimental results demonstrated that variations in valproic acid (VPA) exposure dose and timing significantly impacted neurodevelopmental behavioral indices in male C57BL/6 offspring (Figure 3). The detailed analyses are as follows: (1) Delayed Reflex Development Surface righting reflex (Figure 3D): The number of positive reflex days in the modified group 2 and modified group 1 was significantly lower than that in the control group (P<0.01), whereas no significant difference was observed between the traditional model group (P>0.05). Cliff avoidance reflex (Figure 3A): Compared with the control group, both Modified Groups 1 and 2 presented delayed positive reflex days (P<0.05), with no significant difference between the traditional model group and the control group (P>0.05). Air righting reflex (Figure 3C): Significant delays in positive reflex days were observed in Modified Group 1 and the traditional model group (P<0.05), whereas Modified Group 2 showed a more pronounced delay (P<0.01). (2) Altered Motor Coordination Crawling behavior (Figure 3F): The number of positive reflex days in the modified group 2 and the traditional model group was significantly lower than that in the control group (P<0.05), with modified group 1 displaying a greater delay (P<0.01). In the bar holding test (Figure 3H), Modified Group 2 presented a significant lag in positive reflex days (P<0.01), followed by Modified Group 1 (P<0.05), whereas the traditional model group presented no difference from the control group (P>0.05). (3) Abnormal sensory and stress responses Auditory startle reflex (Figure 3E): The reflex latency in Modified Group 2 was significantly shorter than that in the control group (P<0.001), with a similar trend in Modified Group 1 (P<0.05), suggesting that midgestational VPA exposure may increase auditory sensitivity in offspring. Negative geotaxis reflex (Figure 3B): Modified Groups 1 and 2 presented significant delays in positive reflex days (P<0.01), whereas the traditional model group remained unaffected (P>0.05). Tail-flick photic test reflex (Figure 3G): Latency was significantly prolonged in Modified Group 1 and the traditional model group (P<0.05), with an even greater prolongation in Modified Group 2 (P<0.01). 2.5 Open Field Test Behavioral Analysis The open field test is designed to assess autonomous exploratory behaviors and anxiety-like behavioral traits in experimental animals within a novel environment. The experimental results revealed that valproic acid (VPA) exposure significantly suppressed exploratory behaviors and enhanced anxiety-like responses in C57BL/6 mice (Figure 4). The detailed findings are as follows: 2.5.1 Exploratory behavior Cross center grid (Figure 4A): The number of crossings in Modified Group 1 and the traditional model group was significantly lower than that in the control group (P<0.05), whereas Modified Group 2 exhibited a more pronounced reduction (P<0.01). Inner area distance (Figure 4B): The movement distances in Modified Groups 1 and 2, as well as those in the traditional model group, were significantly lower than those in the control group (P<0.05). 2.5.2 Enhanced Anxiety-like Behaviors Inner area time (Figure 4C): The activity time in Modified Group 1 and the traditional model group was significantly shorter than that in the control group (P<0.05), with Modified Group 2 showing an even greater reduction (P<0.01). Vertical score (Figure 4D): Scores in Modified Group 1 significantly decreased (P<0.05), whereas those in Modified Group 2 substantially decreased (P<0.001). No significant difference was observed between the traditional model group and the control group (P>0.05). 2.6 Three- chamber social behavioral analysis The three-chamber social test was utilized to evaluate social motivation (0–10 min) and social novelty preference (10–20 min) in experimental animals. By measuring exploration time toward unfamiliar conspecifics (Stranger 1/2) versus empty cages/objects, this assay quantified social competence and cognitive flexibility (Figure 5). I. Social Motivation Phase (Socialability, 0–10 min) The experimental results demonstrated that valproic acid (VPA) exposure significantly impaired social interaction in C57BL/6 mice (Figure 5A): (1) Spatial exploration preference Stranger 1 cage duration: Compared with the control group, the traditional model group, modified group 1, and modified group 2 groups presented significantly shorter durations in the Stranger 1 cage (P<0.0001). Empty cage duration: These experimental groups presented a marked increase in time spent in the empty cage (P<0.0001). (2) Social interaction behavior Stranger 1 sniffing time: All VPA-exposed groups presented significantly shorter interaction times with Stranger 1 than did the controls (P<0.0001). Object sniffing time: Exploration time toward objects also notably decreased in the experimental groups (P<0.0001). Conclusion: VPA exposure induced the avoidance of social stimuli (Stranger 1) and a preference for nonsocial environments (empty cages), indicating severe impairment of social motivation (P<0.0001). II. Social novelty preference phase (10- -20 min) Following the introduction of a novel social stimulus (Stranger 2), the behavioral patterns were as follows (Figure 5B): (1) Spatial exploration patterns Stranger 1 cage duration: The experimental groups spent significantly more time in the Stranger 1 cage than the control groups did (P<0.0001). Stranger 2 cage duration: Time in the Stranger 2 cage was dramatically reduced in the experimental groups (P<0.0001). (2) Social interaction disparities Stranger 1 sniffing time: The interaction time with Stranger 1 increased in the experimental groups (P<0.0001). Stranger 2 sniffing time: Exploration of the novel social stimulus (Stranger 2) was significantly diminished (P<0.0001). Conclusion: VPA-exposed mice lacked typical novelty-seeking behavior, suggesting a restricted interest range and compromised social cognitive flexibility.. 2.7 Analysis of repetitive stereotypic behavior (1) Buried marble test The marble-burying test is a classical paradigm for assessing repetitive stereotypic behaviors in rodents. The experimental results demonstrated that VPA exposure significantly increased marble-burying behavior in C57BL/6 mice (Figure 4E). Compared with the control group, Modified Group 2 presented a marked increase in the buried marble count (P<0.01), followed by Modified Group 1 (P<0.05). No significant difference was observed in the traditional model group (P>0.05). (2) Self -grooming VPA-exposed mice displayed excessive self-grooming behavior (Figure 4F): Compared with the control group, the traditional model group, modified Group 1, and modified Group 2 all presented significantly greater grooming frequencies (P<0.05). Conclusion: C57BL/6 mice presented a dose-dependent increase in repetitive stereotypic behaviors following VPA exposure, suggesting potential neurodevelopmental anomalies linked to dysfunction in the basal ganglia-cortical circuit. 2.8 Analysis of Neuroinflammatory Levels in the Prefrontal Cortex ELISAs revealed that valproic acid (VPA) exposure significantly altered the inflammatory cytokine profile in the prefrontal cortex of C57BL/6 mice ( Figure 6 ). The key findings are as follows: (1) Upregulation of proinflammatory cytokines IL-1β (Figure 6A): The IL-1β levels in the Traditional Model Group, Modified Group 1 and Modified Group 2 were significantly greater than those in the Control Group (P<0.05). IL-6 (Figure 6B): IL-6 levels in the traditional model group and modified group 1 were markedly elevated compared with those in the control group (P<0.05), with modified group 2 showing further exacerbation (P<0.01). The TNF-α content (Figure 6C) increased significantly in the traditional model group and modified group 1 (P<0.05), and a similar trend was observed in modified group 2 (P<0.01). (2) Downregulation of anti -inflammatory cytokines IL-10 (Figure 6D): IL-10 levels in the traditional model group and modified group 2 were significantly lower than those in the control group (P<0.05), with modified group 1 demonstrating an even greater reduction (P<0.01). Conclusion: VPA exposure disrupted the proinflammatory/anti-inflammatory balance in the prefrontal cortex of C57BL/6 mice. Modified Group 2 exhibited the most significant enhancement of inflammatory responses, suggesting that its intervention strategy may exacerbate neuroinflammatory processes. 2.9 Analysis of Oxidative Stress Levels in the Prefrontal Cortex ELISAs demonstrated that valproic acid (VPA) exposure significantly disrupted oxidative stress homeostasis in the prefrontal cortex of C57BL/6 mice (Figure 6). The specific findings are as follows: (1) Suppression of Antioxidant Systems Catalase (CAT) (Figure 6E): CAT levels in the traditional model group and modified group 1 were significantly lower than those in the control group (P<0.05), with modified group 2 exhibiting a more pronounced decrease (P<0.01). Glutathione peroxidase (GSH-Px) activity (Figure 6F): GSH-Px activity decreased significantly in the traditional model group and modified group 1 (P<0.05), whereas that in modified group 2 further decreased (P<0.01). Glutathione (GSH) (Figure 6G): The GSH content decreased significantly in the traditional model group and modified group 1 (P<0.05), with a more marked reduction in the modified group 2 (P<0.01). Superoxide Dismutase (SOD) (Figure 6K): SOD levels were significantly lower in Modified Group 1 (P<0.05) and Modified Group 2 (P<0.01), whereas no significant change was observed in the Traditional Model Group (P>0.05). (2) Elevation of Oxidative Damage Markers The malondialdehyde (MDA) content (Figure 6H) was significantly greater in all the experimental groups (traditional model group, modified group 1, and modified group 2) than in the control group (P<0.05). Nitric oxide synthase (NOS) (Figure 6I) and nitric oxide (NO) (Figure 6J): NOS activity and NO levels were significantly elevated in the traditional model group (P<0.05) and modified group 2 (P<0.01), whereas modified group 1 showed no significant changes (P>0.05). Conclusion: VPA exposure induced comprehensive impairment of antioxidant capacity and significant accumulation of oxidative damage markers in the prefrontal cortex of C57BL/6 mice. The modified Group 2 group presented the most severe degree of oxidative stress dysregulation, suggesting that VPA exacerbates redox imbalance in a dose-dependent manner. 2.10 Morris water maze behavioral analysis 2.10.1 Assessment of Learning and Memory Capacity The Morris water maze (MWM) is a cognitively demanding task for rodents that involves complex memory processes. The protocol comprises two phases: spatial acquisition trials and spatial probe trials. Spatial Acquisition Trials During training, the VPA-induced ASD model mice exhibited undirected, random search patterns to locate the hidden platform (Figure 7F). In contrast, control mice demonstrated goal-oriented navigation strategies, with some individuals swimming directly to the platform on the basis of spatial memory (Figure 7G). Compared with control mice, VPA-induced ASD mice presented significantly prolonged escape latencies (P < 0.01; Figure 7C). Spatial Probe Trials Quadrant Residence Time: VPA-induced ASD mice showed no quadrant preference (P > 0.05), whereas control mice spent significantly more time in the target quadrant than in the other quadrants did (P < 0.0001; Figure 7E). Swimming trajectories revealed that control mice predominantly navigated the original platform quadrant, repeatedly crossing the target area (Figure 7I), whereas ASD model mice engaged in aimless exploration across all quadrants (Figure 7H). Platform crossings : The number of platform crossings was significantly lower in VPA-induced ASD mice than in control mice (P < 0.0001; Figure 7A). Target quadrant duration : ASD model mice spent markedly less time in the target quadrant than control mice did (P < 0.0001; Figure 7D), indicating impaired spatial recognition and memory retention. Swimming distance : No significant intergroup differences in total swimming distance were observed (P > 0.05; Figure 7B), confirming preserved motor function in ASD model mice. Conclusion VPA-induced ASD mice exhibited pronounced deficits in spatial learning and memory, as evidenced by prolonged escape latencies, random search strategies, and reduced target quadrant preference. These impairments occurred independently of locomotor dysfunction, underscoring the specificity of cognitive deficits in the ASD model. 2.11 TEM Analysis Reveals Synaptic Ultrastructural Pathologies in VPA-induced ASD Mice Ultrastructural features of the control group Transmission electron microscopy (TEM) analysis revealed characteristic ultrastructural features in the prefrontal cortex (PFC) and hippocampal CA1 region neurons of control mice (Figure 8). The synaptic clefts exhibited narrow spacing, with sharply defined and densely stained postsynaptic densities (PSDs). Synaptic vesicles (SVs) were densely clustered in presynaptic terminals, predominantly adjacent to the presynaptic membrane (Figure 8C). The mitochondria displayed well-organized crista structures with intact membranes and no signs of swelling or shrinkage (Figure 8A). Pathological alterations in the VPA-induced ASD model group (modified group 2) Compared with the control ASD model group, the VPA-exposed ASD model group presented significant synaptic ultrastructural abnormalities (Figure 8D). Presynaptic terminals exhibited marked SV depletion, with the complete absence of SVs in localized areas. Synaptic membrane disorganization manifests as blurred and thickened synaptic clefts, accompanied by indistinct boundaries between pre- and postsynaptic membranes. Notably, abnormal PSD thickening with reduced electron density was observed in select synapses. The mitochondrial pathologies included crista disorganization and membrane rupture, presenting as alternating swelling and shrinkage (Figure 8B). These ultrastructural perturbations indicate impaired SV release at presynaptic terminals and disrupted postsynaptic signaling, potentially altering the excitatory-inhibitory balance. Mitochondrial structural derangements likely exacerbate metabolic insufficiency. Discussion Autism spectrum disorder (ASD) has emerged as a significant global health concern, with its increasing prevalence imposing a heavy burden on families and society[21]. As reported by the US CDC, the prevalence of ASD has reached 1.7%, and in China, it is already 1%[22]. There are nearly 80 million patients worldwide, and this number has surpassed the incidence of childhood tumors, diabetes, leukemia, and AIDS combined[23]. The high cost of caring for approximately one-third of ASD children throughout their lives places substantial economic strain[24]. For example, the annual social cost of ASD in the United States is approximately $236 billion. Given its impact, ASD has become a major public health issue and the leading cause of childhood disability, even being referred to as “mental cancer” with no current cure, sparking extensive research efforts to understand its etiology, as reflected in Science's list of challenging scientific questions. Previous studies have also emphasized the importance of the timing and dose of VPA exposure during pregnancy. Servadio et al. (2018b) reported that the most vulnerable period for VPA-induced pathogenesis in rodents is around embryonic day 12.5, which is a critical period for neural tube closure[15]. Kim et al. (2011) compared VPA exposure at different gestational days in rats and confirmed that exposure at approximately day 12 of pregnancy is most likely to cause offspring morbidity[25]. Williams et al suggested that in humans, exposure to VPA, such as teratogenic chemicals, between the 20th and 24th days of pregnancy, which is equivalent to the 9th and 12th days of pregnancy, is associated with a high incidence of ASD[26]. Most studies use a VPA dose range of 300–800 mg/kg, with 600 mg/kg being a common dose. However, our previous study revealed that a single injection of 600 mg/kg VPA in rats and mice led to high mortality and abortion rates and a low modeling success rate, which is consistent with the findings of other scholars[27]. In this study, we aimed to optimize the establishment of an ASD model in C57BL/6J mice via the use of valproic acid (VPA), addressing the limitations of traditional methods. Our preexperiment, which followed the conventional approach of a single intraperitoneal injection of 600 mg/kg VPA, led to high mortality and abortion rates in pregnant mice, resulting in a low modeling success rate and a significant waste of resources. This finding is consistent with the understanding that high-dose VPA has strong toxic side effects. The innovative aspect of our study lies in the administration method. Conventionally, mice and rats are mated at 17:00, and pregnancy is checked at approximately 9:00 the next morning, with a 16-hour interval during which the actual fertilization time varies greatly among individuals. The traditional literature typically calculates the fertilization time as approximately 2:00 am for determining the 12.5-day dosing time, but this is inaccurate. Single-dose administration can ensure accurate dosing for only approximately 1/3 of the animals, and two-dose administration can achieve only 2/3 accuracy. In contrast, our novel three-dose administration strategy addresses this issue. By administering three doses of VPA at different times around E12.5 (for example, in C57BL/6 mice, the modified model 1 group received doses of 200 mg/kg on E12, 300 mg/kg on E12.5, and 200 mg/kg on E13; the modified model 2 group received 300 mg/kg on E11.5, 400 mg/kg on E12.5, and 300 mg/kg on E13.5), we can ensure that all the animals receive the drug at the most accurate time, regardless of whether they conceive earlier or later. This significantly improves the modeling efficiency, reduces experimental costs, minimizes side effects, and aligns better with animal welfare principles. To further increase the precision of the dosing time, several additional strategies could be considered. One approach could be to use more advanced detection techniques to determine the exact time of fertilization. For example, noninvasive imaging methods or hormonal assays could be employed to detect early signs of fertilization more accurately. By closely monitoring hormonal changes in female animals, such as the levels of progesterone or luteinizing hormone, it may be possible to pinpoint the time of ovulation and subsequent fertilization more precisely. This would allow for a more individualized dosing schedule tailored to the specific fertilization time of each animal. Our modified method not only reduces the side effects on pregnant mice but also better mimics the clinical situation of pregnant women taking VPA-related drugs multiple times. The traditional single-injection method caused severe acute toxicity in pregnant C57BL/6 mice, with a 16.7% mortality rate within 6 h and a 50% abortion rate. In contrast, the modified groups had no mortality, and their abortion rates were significantly lower. This is because multidose administration allows the body to adapt to the drug gradually, and the drug remains in the body for a longer and more stable period, which may cause more severe damage to the brain development of offspring in a more physiological way. The offspring of the mice in the modified groups also exhibited more prominent ASD-related behaviors. In behavioral tests such as the open-field test, three-chamber social test, and self-grooming test, the male offspring of C57BL/6 mice presented characteristics similar to those of ASD patients, such as reduced exploration, social deficits, and increased repetitive behaviors. Compared with other models, our improved ASD model results in a significantly greater delivery rate to pregnant mice and a greater rate of external malformations in newborn pups. These findings indicate that our method can more effectively simulate the core symptoms of ASD, greatly improving the modeling efficiency, reducing experimental costs, and protecting animal welfare. In conclusion, our optimized modeling method provides a more reliable and efficient tool for studying the pathogenesis of ASD and developing potential therapeutic strategies, which may contribute to the ultimate conquest of this disorder. Summary: Optimized Maternal Safety: The phased dosing regimen significantly improved maternal survival rates and reduced abortion rates, resolving acute toxicity challenges . Robust ASD-like phenotypes : Offspring exhibit core behavioral deficits, including impaired social interaction, heightened repetitive behaviors and spatial memory dysfunction. Neurobiological correlates : Synaptic ultrastructural abnormalities (presynaptic vesicle depletion, mitochondrial crista disorganization) and dysregulation. Cost efficiency : The protocol reduces costs while maintaining phenotypic consistency, increasing accessibility for resource-limited laboratories. Research Contributions. Methodological innovation : This study pioneers chronopharmacological ASD modeling, aligning VPA exposure with critical neurodevelopmental windows to improve temporal precision. Ethical and Practical Advancements: By minimizing maternal toxicity and teratogenicity, the protocol adheres to the \"3R\" principles (reduction, refinement, replacement), setting a new standard for ethical animal research. Resource Optimization: Laboratories in developing regions can leverage cost-saving designs to scale ASD research without compromising quality. Limitations and Future Directions: Model generalizability : Current validation is limited to C57 mice; future studies should test the protocol in rats and nonhuman primates to assess cross-species applicability. Integration with multiomics : Combining this model with single-cell RNA sequencing or metabolomics could uncover novel biomarkers and mechanistic pathways. Acknowledgments: This work was supported by grants from the Enterprise Joint Fund Project of Hunan Provincial Natural Science Foundation, 2024JJ9097; the Enterprise Fund Project supported by ZYYK; and the GDAS Project of Science and Technology Development (2022GDASZH-2022010110, 2022GDASZH-2022030603-01, 2023GDASZH-2023030602). Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors’ contributions ZML, CXW and ZYL conceived and designed the study; ZML, CXW, ZXL, LF and HYL performed the experiments; ZML and CXW analyzed the data; ZML and CXW visualized the figures; ZML and CXW wrote the manuscript draft; ZML, CXW and ZYL revised the manuscript and supervised the study; all the authors contributed to the article and approved the submitted version. Data availability All datasets generated and analyzed during this study are presented in this published article. Competing Interests The authors declare that they have no competing interests. Ethics approval Animal care and experimental procedures were performed in accordance with the institutional laboratory animal guidelines. The protocols for the animal experiments were as follows: approved by the Institutional Animal Care and Use Committee of Guangzhou Institutes of Biomedicine and Health, CAS (IACUC:2023081). References Kutluk, G., Kadem, N., Bektas, O. & Eroglu, H. N. 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Li\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3OMQrCMBSA4Vce1CXYVRD1ChFBBC9jEHRR6AmkUNBFcC0IegVdxLGSwaXuGRwUQRyVTIKDiQpubUbB/MMjhHxJAGy2HyyPAAhULzEGFsTZxP0St2VI9PXvJaHgGJEcoPR9zibTsbwc13vwRrEj/YyP1SLKWbTfrRosOUMhaWExyiBtokgg+ivKhhxAqE2SQbgmc9E7v0jFgDihJgvRw4Mm1IAgEtqtLUWnDizhpJqwsJhGPC9GSR7N0ky0T7f7mpfLW76RaUShqxrh68WCGuqwE6SCTwM98Gpy1Gaz2f6vJx4fSZgjMiTVAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Zhiyuan\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-04-28 08:38:05\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6545657/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6545657/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":89054096,\"identity\":\"ece9ed73-44c2-4063-8e1d-3b59d19d1d18\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:11:19\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14075832,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eConstruction and phenotypic characterization of ASD models in C57BL/6 mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) establishment of the ASD model in a neonatal C57BL/6 mouse (modified protocol); (B) taillength measurement in the C57BL/6 mouse ASD model; (C) Classic tail curvature phenotype in the C57BL/6 mouse ASD model\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/1a01708ff992a6ad369cecb1.png\"},{\"id\":89054111,\"identity\":\"28be237d-59d0-465f-845c-309d08f28eea\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:11:20\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1558374,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDose- and time-dependent developmental toxicity of VPA exposure in a C57BL/6 mouse ASD model\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Body mass development curve. (B) Tail length growth curves. (C) Age of positive incisor eruption. (D) Age of positive fur development. (E) Age of positive eye opening. The data are presented as the means ± SDs (x̅±s, n=12). Statistical significance: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001, compared with the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/0dc1505be22b0b5450fa58eb.png\"},{\"id\":89053044,\"identity\":\"76e5d1ce-f579-40ec-a984-fc4fc9fb9814\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:20\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2031112,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eEffects of VPA Exposure at Different Doses and Time Points on Neurological Development in Male Offspring\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Cliff avoidance reflex (B) Negative geotaxis reflex (C) Air righting reflex (D) Surface righting reflex (E) Auditory startle reflex (F) Crawling (G) Tail-flick photic test reflex (H) Bar holding test. The data are presented as the means ± SDs (x̅±s, n=12). Statistical significance: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001, compared with the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/03e8d3a6d73fc746a4c12ddc.png\"},{\"id\":89053057,\"identity\":\"6fcc720b-f0d7-4d8d-a04e-295e10b0d6e5\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:20\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":8363474,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBehavioral Analysis of the Open Field and Repetitive Stereotypic Behavior of C57BL/6 Mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Cross-center grid; (B) innerarea distance; (C) inner area time; (D) vertical score; (E) buried marble histogram; (F) self-grooming; (G) buried marbles; (H) open field trajectory diagram of the ASD model; (I) open field trajectory map of the control group. The data are presented as the means ± SDs (x̅±s, n=12). Statistical significance: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001, compared with the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/c72a7b89241cad0009c8598d.png\"},{\"id\":89053034,\"identity\":\"c0eefeb0-b3db-4fbb-bf74-93bf601bd9f0\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:19\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":7954331,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBehavioral analysis of three-chamber social behaviors in C57BL/6 mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Social ability; (B) \\u003cstrong\\u003esocial novelty\\u003c/strong\\u003e; (C) trajectory diagram of social ability (0--10 min) in the ASD model group; (D) trajectory map of socialability (0--10 min) in the control group; (E) trajectory diagram of the social novelty preference phase (10--20 minutes) in the ASD model group; (F) trajectory diagram of the social novelty preference phase (10--20 minutes) in the control group. The data are presented as the means ± SDs (x̅±s, n=12). Statistical significance: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001, compared with the control group. Stranger 1: timespent in the stranger 1 cage; object cage: timespent in the object cage; stranger 1: interaction time with stranger 1; object: interaction time with the object; stranger 2: time spent in the stranger 2 cage; stranger 2: interaction time with stranger 2.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/d1935724638400f14b01d3da.png\"},{\"id\":89053062,\"identity\":\"6ec6dbab-3888-498e-bae7-a9d67de99894\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:21\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2714366,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnalysis of neuroinflammatory and oxidative stress levels in the prefrontal cortex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) IL-1β (B) IL-6 (C) TNF-α (D) IL-10 (E) CAT content; (F) GSH-Px activity; (G) GSH content; (H) SOD activity; (I) MDA content; (J) NOS activity; (K) NO level. The data are presented as the means ± SDs (x̅±s, n=12). Statistical significance: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001, compared with the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/232ac6c24c75ddfae8490b7c.png\"},{\"id\":89054115,\"identity\":\"3519c33d-684d-4b98-ba72-698cb602a9cd\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:11:20\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2098277,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eBehavioral analysis of the Morris water maze\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Number of platform crossings. (B) Total swimming distance. (C) Escape latency to locate the hidden platform. (D) Time spent in the target quadrant. (E) Time distribution across the four quadrants during the spatial probe trial. (F) Representative swimming paths of VPA-induced ASD model mice during spatial acquisition trials. (G) Swimming paths of control mice during spatial acquisition trials. (H) Exploration trajectories of ASD model mice during spatial probe trials. (I) Exploration trajectories of control mice during spatial probe trials. The data are presented as the means ± SDs (x̅±s, n=12). Statistical significance: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001, compared with the control group.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/254d11a670ad5e53ac6d3467.png\"},{\"id\":89053060,\"identity\":\"36042f2e-e3e1-4d8b-a66f-7d9dff033ef4\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:21\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":41676979,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCharacteristic ultrastructural features in the prefrontal cortex (PFC) and hippocampal CA1 region neurons of control and ASD model mice.\\u003c/p\\u003e\\n\\u003cp\\u003e(A) Representative ultrastructural features of mitochondria in the control group. (B) Mitochondrial ultrastructural characteristics of the ASD model group. (C) Synaptic ultrastructure in the control group. (D) Synaptic ultrastructural morphology in the ASD model group. Representative pictures from n = 3 independent experiments for the control and ASD model animals are presented.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/41db33b8ec344f1fa7cff33a.png\"},{\"id\":106403013,\"identity\":\"7b3dd724-93dc-49b7-8141-5ee6e5578ef8\",\"added_by\":\"auto\",\"created_at\":\"2026-04-08 09:13:23\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":87725566,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/ea7bd48e-87ae-4e4c-a7ee-68e6f8672dcd.pdf\"},{\"id\":89053027,\"identity\":\"94c2fae0-e4f3-4fda-8428-bb7a95a1626c\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:19\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":14447,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/254279aae2ef0b93cf590ed4.docx\"},{\"id\":89053031,\"identity\":\"e371f8af-0f44-4085-b9dc-7f2f9e42a902\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:19\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":14603,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table2.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/5c48ac667cd7a5dcb95af6f8.docx\"},{\"id\":89053032,\"identity\":\"a392b801-2f0e-49e8-8662-be05ac6adbce\",\"added_by\":\"auto\",\"created_at\":\"2025-08-14 08:03:19\",\"extension\":\"pdf\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":469467,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"AnimalExperimentWelfareandEthicsapprovaldocument.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6545657/v1/28194333e5ee5f5531273550.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Triple-Phase VPA Administration in C57 Mice: A Precision Timing Strategy for Cost-Effective ASD Modeling with Reduced Maternal Toxicity\",\"fulltext\":[{\"header\":\"Key Highlights\",\"content\":\"\\u003cp\\u003eThis article presents the first application of chronopharmacological principles to ASD mouse models via phase-specific VPA dosing.\\u003c/p\\u003e\\n\\u003cp\\u003e100% maternal survival with significant cost reduction, addressing Nature Protocols' call for affordable models.\\u003c/p\\u003e\\n\\u003cp\\u003ePhenotypic consistency has improved, addressing reproducibility challenges in existing models.\\u003c/p\\u003e\\n\\u003cp\\u003eBehavioral, ultrastructural, and molecular profiling can be combined to confirm the ASD-like pathophysiology.\\u003c/p\\u003e\\n\\u003cp\\u003eMaternal toxicity and embryotoxicity were mitigated in accordance with the \\\"3R\\\" principles (reduction, refinement, replacement).\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eAutism spectrum disorder (ASD) is a neurodevelopmental condition characterized by persistent deficits in social communication and interaction alongside restricted and repetitive behaviors, affecting approximately 1\\u0026ndash;2% of the global population [1, 2]. Despite decades of research, the etiology of ASD remains elusive, with heterogeneous genetic and environmental contributions complicating\\u0026nbsp;the\\u0026nbsp;mechanistic understanding\\u0026nbsp;[3]. Animal models, particularly rodents, have become indispensable for studying ASD pathophysiology, yet existing paradigms face critical limitations in\\u0026nbsp;terms of\\u0026nbsp;biological validity, reproducibility, and ethical feasibility\\u0026nbsp;[4]. This study introduces a novel triple-phase valproic acid (VPA) administration protocol in\\u0026nbsp;C57BL/6J\\u0026nbsp;mice\\u0026nbsp;that is\\u0026nbsp;designed to overcome these challenges through precision timing,\\u0026nbsp;reduce\\u0026nbsp;maternal toxicity, and\\u0026nbsp;increase\\u0026nbsp;cost-effectiveness.\\u003c/p\\u003e\\n\\u003cp\\u003eASD encompasses a wide range of conditions with significant heterogeneity in symptoms and severity [5]. The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) defines ASD on\\u0026nbsp;the basis of\\u0026nbsp;deficits in social communication and restricted, repetitive behaviors\\u0026nbsp;[6]. Children with ASD often\\u0026nbsp;face\\u0026nbsp;challenges in social interactions, such as difficulties in making eye contact, understanding social cues, and engaging in reciprocal conversations\\u0026nbsp;[7]. They may also exhibit repetitive behaviors\\u0026nbsp;such as hand-flapping, rocking, or insistence on sameness\\u0026nbsp;[8]. These symptoms can significantly impact their daily lives, education, and future social and occupational functioning\\u0026nbsp;[9].\\u003c/p\\u003e\\n\\u003cp\\u003eAnimal models, especially rodent models, are invaluable tools for studying the pathophysiology of ASD and evaluating potential therapeutic interventions [10]. Mice and rats are commonly used\\u0026nbsp;because of\\u0026nbsp;their genetic similarity to humans, short reproductive cycles, and ease of genetic manipulation\\u0026nbsp;[11]. There are various types of rodent models for ASD, including genetic models and neurotoxicological models\\u0026nbsp;[12]. Genetic models, such as Shank3 knockout mice and BTBR T+tf/J mice, mimic specific genetic mutations associated with ASD in humans and display behavioral deficits similar to those\\u0026nbsp;observed\\u0026nbsp;in ASD patients\\u0026nbsp;[13]. Neurotoxicological models,\\u0026nbsp;such as\\u0026nbsp;the VPA-induced model, are generated by exposing pregnant rodents to VPA, leading to offspring with ASD-like behaviors\\u0026nbsp;[14].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLimitations of Current Models\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(1) Temporal and Dose Inaccuracy\\u003c/p\\u003e\\n\\u003cp\\u003eCritical neurodevelopmental windows (e.g., neural tube closure at E11.5\\u0026ndash;E13.5) require precise VPA timing, but individual variability in conception complicates single-dose synchronization. Melancia et al. (2018) demonstrated that only 33% of litters receive VPA during optimal windows, leading to\\u0026nbsp;interlitter\\u0026nbsp;variability (CV \\u0026gt; 30%) in cortical migration deficits [15].\\u003c/p\\u003e\\n\\u003cp\\u003e(2)\\u0026nbsp;Maternal Toxicity and Ethical Concerns\\u003c/p\\u003e\\n\\u003cp\\u003eHigh-dose VPA triggers placental insufficiency and metabolic acidosis, resulting in abortion rates exceeding 50% [14]. Such outcomes\\u0026nbsp;contradict\\u0026nbsp;the \\u0026quot;3R\\u0026quot; principles (replacement, reduction, refinement), limiting ethical approval and scalability\\u0026nbsp;[16].\\u003c/p\\u003e\\n\\u003cp\\u003e(3) Cost-effectiveness and\\u0026nbsp;generalizability\\u003c/p\\u003e\\n\\u003cp\\u003eThe cost of establishing and maintaining rodent models for ASD research can be high, especially for genetic models that require complex genetic engineering techniques [17]. This may limit the widespread use of these models, particularly in resource-limited laboratories. Additionally, the generalizability of the findings from rodent models to humans is still a matter of debate. Therefore, it is essential to develop more cost-effective and translatable rodent models to improve the relevance of the research findings.\\u003c/p\\u003e\\n\\u003cp\\u003eThe core objective of this study\\u0026nbsp;was\\u0026nbsp;to develop an optimized, cost-effective C57 mouse model of ASD that can accurately recapitulate the behavioral and pathophysiological features of the disorder.\\u0026nbsp;On the basis of\\u0026nbsp;previous research, we hypothesize that a triple-phase VPA regimen (300\\u0026rarr;400\\u0026rarr;300 mg/kg at E11.5, E12.5,\\u0026nbsp;and\\u0026nbsp;E13.5) will\\u0026nbsp;increase maternal survival: distributing\\u0026nbsp;doses across gestational stages reduces peak plasma concentrations, mitigating acute toxicity.\\u0026nbsp;Optimize Neurodevelopmental Targeting: Ensure\\u0026nbsp;that\\u0026nbsp;all litters receive VPA during E11.5\\u0026ndash;E13.5, irrespective of conception timing.\\u0026nbsp;Maintaining\\u0026nbsp;Phenotypic Fidelity:\\u0026nbsp;Preserving\\u0026nbsp;ASD-like behaviors and synaptic pathologies while lowering cumulative VPA exposure.\\u003c/p\\u003e\\n\\u003cp\\u003eTo address these challenges, we developed a triple-phase VPA administration protocol (300\\u0026rarr;400\\u0026rarr;300 mg/kg at E11.5, E12.5, and E13.5) that integrates chronopharmacological principles with neurodevelopmental staging. Our protocol introduces three transformative elements:\\u003c/p\\u003e\\n\\u003cp\\u003eChronopharmacological\\u0026nbsp;alignment: The phased regimen ensures\\u0026nbsp;that\\u0026nbsp;all offspring receive VPA during E11.5\\u0026ndash;E13.5, regardless of conception timing\\u0026mdash;a critical advancement over single-dose methods.\\u003c/p\\u003e\\n\\u003cp\\u003eToxicity\\u0026nbsp;mitigation: By avoiding\\u0026nbsp;suprathreshold\\u0026nbsp;VPA concentrations (IC50 for embryotoxicity: 450 mg/kg), we reduce mitochondrial dysfunction in placental trophoblasts, a key driver of pregnancy loss.\\u003c/p\\u003e\\n\\u003cp\\u003eMultidimensional Validation: Combining automated behavioral tracking (EthoVision XT), synaptic ultrastructural analysis (TEM), and neuroinflammatory profiling to establish causal links between molecular perturbations and ASD-like behaviors.\\u003c/p\\u003e\\n\\u003cp\\u003ePrior studies have laid essential groundwork but lack translational cohesion. For example, Nicolini and Fahnestock established the role of VPA in altering the GABA/glutamate balance[18], whereas Roullet et al. linked prenatal VPA exposure to prefrontal‒amygdala circuit hyperconnectivity[19]. However, neither the inherent toxicity nor the reproducibility of the protocol has been addressed. Our work directly builds on these findings by introducing a timing-optimized model that preserves synaptic pathologies (e.g., vesicle depletion, PSD thickening) while eliminating confounding maternal mortality. Furthermore, we extended the mechanistic scope by demonstrating dose-dependent neuroinflammatory dysregulation, a feature that is absent in genetic models such as Shank3 knockouts[20].\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e1.1\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eExperimental Materials\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cbr\\u003e1.1.1\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Experimental Animals\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAdult C57BL/6 mice (96 females, 48 males, body weights of 20\\u0026ndash;30 g) were purchased from Zhuhai Baite Tong Biotechnology Co., Ltd. (Production License Number: SCKK (Yue) 2020\\u0026ndash;0051; C57BL/6 mouse certificate number: 44822700030567). All animals were housed in the SPF-level animal facility of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Housing License Number: SYXK (Yue) 2022\\u0026ndash;0063), under controlled environmental conditions: temperature 22\\u0026deg;C \\u0026plusmn; 2\\u0026deg;C, relative humidity 50%\\u0026ndash;60%, 12 h/12 h light\\u0026ndash;dark cycle, with ad libitum access to standard rodent diet and sterilized drinking water.\\u003c/p\\u003e\\n\\u003cp\\u003eThis study is reported in accordance with the ARRIVE guidelines (Percie du Sert et al., 2020). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Ethics Approval No. CAS (IACUC:2023081)), and strictly adhered to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 8th edition, 2011). Euthanasia and anesthesia protocols followed the 2020 American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, and no prohibited methods (e.g., chloral hydrate, ether, or chloroform) were employed. Every effort was made to minimize animal suffering, including the use of appropriate analgesic regimens and humane endpoints during the study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.1.2\\u003c/strong\\u003e\\u003cstrong\\u003eReagents and Equipment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eValproic acid (VPA, Sigma‒Aldrich Company, USA)\\u003cstrong\\u003e\\u0026nbsp;was used.\\u003cbr\\u003eThe behavioral\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;testing system\\u0026nbsp;\\u003c/strong\\u003eincluded a \\u0026nbsp;plane righting reflex test platform (20\\u0026times;20\\u0026times;2 cm), an auditory startle reflex device (1 cm\\u0026sup3; stainless steel block placed on a 35\\u0026times;30\\u0026times;0.5 cm metal plate), three chamber test boxes (60\\u0026times;40\\u0026times;22 cm), and an open field test box (50\\u0026times;50\\u0026times;30 cm).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.2\\u003c/strong\\u003e\\u003cstrong\\u003ePreparation of\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ethe\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eASD\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eanimal model induced\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;by VPA\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAdult male and female C57BL/6 mice were placed in the same cage at 17:00 p.m. in the SPF animal room of\\u0026nbsp;the\\u0026nbsp;Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. On the 2nd day after cage placement, vaginal checks were conducted at 9:00 a.m. The presence of vaginal plugs was considered successful fertilization, and the date of vaginal plug appearance was recorded as the 0.5th day of pregnancy.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.2.1 C57BL/6 Mice\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ewere randomly divided\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;into 4 groups\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;according to the method of VPA administration\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(1) Traditional Model Group:\\u003c/p\\u003e\\n\\u003cp\\u003eE12.5 injection of VPA (valproic acid sodium) (600 mg/kg)\\u003c/p\\u003e\\n\\u003cp\\u003e(2) Modified Model Group 1:\\u003c/p\\u003e\\n\\u003cp\\u003eMultiple administrations (E12: 200; E12.5: 300; E13:\\u0026nbsp;200 mg/kg)\\u003c/p\\u003e\\n\\u003cp\\u003e(3) Modified Model Group 2:\\u003c/p\\u003e\\n\\u003cp\\u003eMultiple administrations (E11.5: 300; E12.5: 400; E13.5:\\u0026nbsp;300 mg/kg)\\u003c/p\\u003e\\n\\u003cp\\u003e(4) Control\\u0026nbsp;group: E12.5 injection of\\u0026nbsp;an\\u0026nbsp;equal volume of normal saline\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.2.3\\u003c/strong\\u003e \\u003cstrong\\u003eObservation\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eindices\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;after\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003egroup administration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e①\\u0026nbsp;Observe the adverse reactions,\\u0026nbsp;mortality,\\u0026nbsp;and\\u0026nbsp;abortion\\u0026nbsp;rates\\u0026nbsp;of pregnant mothers in each group after\\u0026nbsp;the\\u0026nbsp;administration of valproic acid sodium or normal saline;\\u003c/p\\u003e\\n\\u003cp\\u003e②\\u0026nbsp;Observe the morphology,\\u0026nbsp;growth and development of newborn pups in each group;\\u003c/p\\u003e\\n\\u003cp\\u003e③\\u0026nbsp;The\\u0026nbsp;social behavior of newborn pups in each group\\u0026nbsp;was observed.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.3 Reproductive\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eoutcome analysis in each group\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e①\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Delivery rate assessment:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe number of pregnant C57 mice that\\u0026nbsp;were\\u0026nbsp;successfully delivered was recorded for each experimental group.\\u0026nbsp;The delivery\\u0026nbsp;rate was calculated as\\u0026nbsp;follows:\\u003c/p\\u003e\\n\\u003cp\\u003eDelivery rate (%) =\\u0026nbsp;number of delivered mice/number of pregnant mice \\u0026times; 100%.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e②\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Neonatal morphological abnormality evaluation:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe\\u0026nbsp;appearance of the skull and limbs of pups (including male and female pups) in each group\\u0026nbsp;was observed\\u0026nbsp;on the day of birth, and\\u0026nbsp;the presence or absence of appearance deformities (i.e., short development of limbs, inconspicuous eyeballs and auricles) of pups in each group (except for tail deformities)\\u0026nbsp;was determined.\\u0026nbsp;The malformation rate was determined as\\u0026nbsp;follows:\\u0026nbsp;Malformation rate (%) = (number\\u0026nbsp;of malformed pups/total\\u0026nbsp;live births) \\u0026times;100%.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.4\\u003c/strong\\u003e\\u003cstrong\\u003eAdverse\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ereactions\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003emortality in pregnant mice following drug administration\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter intraperitoneal (i.p.) injection, pregnant mice were returned to their home cages. Adverse reactions were monitored for 30 min\\u0026nbsp;postinjection, and mortality was recorded within 6 h.\\u0026nbsp;The results\\u0026nbsp;are\\u0026nbsp;summarized in the following table.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.5\\u003c/strong\\u003e\\u003cstrong\\u003eAbortion\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003erate analysis in pregnant mice after treatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAbortion was determined on the\\u0026nbsp;basis of the\\u0026nbsp;following criteria:\\u003c/p\\u003e\\n\\u003cp\\u003e①\\u0026nbsp;Vaginal plug presence\\u0026nbsp;was\\u0026nbsp;confirmed at 9:00 AM the morning after mating.\\u003c/p\\u003e\\n\\u003cp\\u003e②\\u0026nbsp;Significant body weight gain and abdominal distension\\u0026nbsp;were detected\\u0026nbsp;by gestational day (GD) 12.5.\\u003c/p\\u003e\\n\\u003cp\\u003e③\\u0026nbsp;Sudden body weight loss between\\u0026nbsp;GDs\\u0026nbsp;19\\u0026ndash;20.\\u003c/p\\u003e\\n\\u003cp\\u003ePregnant mice fulfilling all three criteria were classified as aborted.\\u003c/p\\u003e\\n\\u003cp\\u003eThe abortion rate (%) was calculated as\\u0026nbsp;the\\u0026nbsp;number of aborted females/number of pregnant females in each group\\u0026times;100%.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.6\\u003c/strong\\u003e\\u003cstrong\\u003eEffects of VPA Exposure on\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ethe\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ePhysiological Development\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eof\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Male Offspring\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e(1) Four-day survival rate of male pups:\\u003c/p\\u003e\\n\\u003cp\\u003e4-day survival rate (%) = (Male pups surviving at postnatal day 4/Live male births) \\u0026times;100%\\u003c/p\\u003e\\n\\u003cp\\u003e(2) Body\\u0026nbsp;mass:\\u0026nbsp;Body mass was measured via an electronic balance (each animal was measured three times, and the average was calculated).\\u003c/p\\u003e\\n\\u003cp\\u003e(3) Tail\\u0026nbsp;length:\\u0026nbsp;The length was measured\\u0026nbsp;from\\u0026nbsp;the\\u0026nbsp;tail base to\\u0026nbsp;the\\u0026nbsp;tip in\\u0026nbsp;the\\u0026nbsp;extended position (each animal was measured three times, and the average\\u0026nbsp;length\\u0026nbsp;was calculated).\\u003c/p\\u003e\\n\\u003cp\\u003e(4) Tail\\u0026nbsp;curvature evaluation (PND 30):\\u003c/p\\u003e\\n\\u003cp\\u003eTail curvature rate (%) = (Adult males with tail curvature/Total adult males) \\u0026times;100%\\u003c/p\\u003e\\n\\u003cp\\u003e(5) Incisor eruption: Positive when lower incisors exhibited visible white spots and tactile resistance\\u003c/p\\u003e\\n\\u003cp\\u003e(6) Eye opening: Positive when \\u0026ge;50% of the orbital area is exposed\\u003c/p\\u003e\\n\\u003cp\\u003e(7) Fur development: Positive when abdominal skin pigmentation changes from pink to black (C57 mice) with a fur coat.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.7\\u003c/strong\\u003e\\u003cstrong\\u003eEffects of VPA Exposure on Neurological Development in Male Offspring\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe neurological development of male pups was evaluated via four standardized reflex tests: surface righting reflex, cliff avoidance reflex, air righting reflex, and pivoting behavior. The postnatal age at which each reflex reached positive criteria was recorded for all pups in each group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Surface\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Righting Reflex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProcedure: Pups were placed in a supine position on a flat surface for 3 sec and released.\\u003c/p\\u003e\\n\\u003cp\\u003ePositive criterion: Complete rotation to\\u0026nbsp;the\\u0026nbsp;prone position with all four paws contacting the surface within 10 sec.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Cliff avoidance\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Reflex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProcedure: Pups were positioned at the edge of a 30 cm-high platform with their heads protruding.\\u003c/p\\u003e\\n\\u003cp\\u003ePositive criterion: Turning or retreating movement within 10 sec.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(3) Air righting reflex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProcedure: Pups were held supine 30 cm above a soft cushion for 3 sec before release.\\u003c/p\\u003e\\n\\u003cp\\u003ePositive criterion: Successful landing in\\u0026nbsp;the\\u0026nbsp;prone position in 3 consecutive trials.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(4) Negative\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Geotaxis Reflex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProcedure: Pups were placed head-down on a 25\\u0026deg; inclined plane (PND 7--10).\\u003c/p\\u003e\\n\\u003cp\\u003ePositive criterion: Completion of 180\\u0026deg;\\u0026nbsp;turning\\u0026nbsp;to\\u0026nbsp;the\\u0026nbsp;head-up position within 30 sec.\\u003c/p\\u003e\\n\\u003cp\\u003eFunctional significance: This test evaluates vestibular function and motor coordination.\\u0026nbsp;A prolonged\\u0026nbsp;turning time may indicate impaired vestibular development.\\u003c/p\\u003e\\n\\u003cp\\u003eNote: All reflex tests were performed daily until all pups in each group met the\\u003c/p\\u003e\\n\\u003cp\\u003epositive\\u0026nbsp;criterion. The developmental age for each reflex was recorded when 100%\\u003c/p\\u003e\\n\\u003cp\\u003ePups\\u0026nbsp;in a group achieved\\u0026nbsp;a\\u0026nbsp;positive response.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(5) Bar\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Holding Test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProcedure: Pups were suspended by\\u0026nbsp;their\\u0026nbsp;forelimbs on a horizontal bar (25 cm height).\\u003c/p\\u003e\\n\\u003cp\\u003ePositive criterion: Maintaining grip for \\u0026ge;2 seconds\\u003c/p\\u003e\\n\\u003cp\\u003eSignificance: Evaluates forelimb strength and grip endurance\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(6) Crawling behavior\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProcedure: Pups were placed on a flat surface\\u003c/p\\u003e\\n\\u003cp\\u003ePositive criterion: Independent forward locomotion (\\u0026ge;3 cm) using all four limbs while maintaining ventral contact with\\u0026nbsp;the\\u0026nbsp;surface\\u003c/p\\u003e\\n\\u003cp\\u003eSignificance:\\u0026nbsp;Assessment of\\u0026nbsp;early locomotor coordination\\u003c/p\\u003e\\n\\u003cp\\u003eThe postnatal age at which 100% of\\u0026nbsp;the\\u0026nbsp;pups in each group achieved positive responses was recorded for both tests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(7) Auditory startle reflex assessment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe auditory startle reflex was evaluated daily from postnatal day (PND) 2 using a standardized protocol:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTest Procedure:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA metal plate was positioned horizontally 15 cm below the test subject.\\u0026nbsp;A metal block was dropped vertically onto the plate to generate an abrupt acoustic stimulus (90\\u0026ndash;100\\u0026nbsp;dB).\\u0026nbsp;Testing was conducted in a sound-attenuated chamber.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePositive\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ecriterion\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eImmediate whole-body startle response (distinct curling or trembling) following sound presentation.\\u0026nbsp;The developmental milestone was recorded as the postnatal age when 100% of\\u0026nbsp;the\\u0026nbsp;male pups in each group exhibited consistent positive responses.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(8)\\u0026nbsp;Tail\\u0026ndash;Flick\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Analgesia Test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe tail-flick test assesses nociceptive responses in rodents by measuring withdrawal latency to radiant heat stimulation. This method offers distinct advantages over hot plate testing, particularly its applicability to lightly anesthetized animals and independence from motor coordination.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTesting Procedure:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e①\\u0026nbsp;The\\u0026nbsp;experimental animals\\u0026nbsp;were removed\\u0026nbsp;from the animal room,\\u0026nbsp;weighed, allowed to\\u0026nbsp;acclimatize in the laboratory for 30 min, and the\\u0026nbsp;control\\u0026nbsp;group\\u0026nbsp;was separated\\u0026nbsp;from the model group.\\u003c/p\\u003e\\n\\u003cp\\u003e②\\u0026nbsp;Determine the baseline latencies of the animals with a tail flash tester (i.e., there is a heat source under the plate of a small hole), put the tail of the mouse (approximately 50 mm\\u0026nbsp;in front of the tail tip) or mouse (approximately 15 mm\\u0026nbsp;in front of the tail tip) on top of the small hole, start the heat source to start timing until the tail dodges, adjust the intensity of the light source, set most of the time of the tail dodge time\\u0026nbsp;to 3\\u0026ndash;4 s;\\u0026nbsp;if there is no dodge reflex,\\u0026nbsp;then set the test termination time to\\u0026nbsp;10\\u0026nbsp;s\\u0026nbsp;to avoid burns;\\u003c/p\\u003e\\n\\u003cp\\u003e③\\u0026nbsp;Test the tail flash reaction, put the animal on the tail flash test board and put its tail on the small hole of the light source, that is, start timing, observe the time of the animal\\u0026apos;s tail dodging reaction, or until the termination (cutoff) time. Note that only 1 time point is measured in a test, not 3.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.8\\u003c/strong\\u003e\\u003cstrong\\u003eBehavioral Assessments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA researcher, blinded to the group assignments, conducted the behavioral evaluations. ASD-like behavior was scored\\u0026nbsp;via Noldus EthoVision XT software (Noldus, Netherlands). The behavioral assessments were carried out during the day between 09:00 and 18:00.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.8.1\\u003c/strong\\u003e\\u003cstrong\\u003eOpen\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003efield test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe open field test\\u0026nbsp;was used to evaluate\\u0026nbsp;exploratory behavior and anxiety-like responses in rodents exposed to a novel environment. This paradigm exploits the natural conflict between rodents\\u0026apos; exploratory drive and their aversion to open spaces.\\u003c/p\\u003e\\n\\u003cp\\u003eAn overhead camera system was used to record the path, and Noldus EthoVision XT software was employed to determine the frequency of entries and the time duration within the central area. The open field test (OFT) is a common assay for evaluating both anxiety-like and motor behaviors in animals (Kraeuter et al., 2019).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eProtocol:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ea) Habituation (10 min): Animals freely explored the apparatus\\u003c/p\\u003e\\n\\u003cp\\u003eb) Testing (10 min): Recorded parameters included\\u0026nbsp;the following:\\u003c/p\\u003e\\n\\u003cp\\u003eCentral/peripheral crossings (all limbs crossing sector borders)\\u003c/p\\u003e\\n\\u003cp\\u003eVertical activity (forelimbs raised \\u0026ge;2 s)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnalysis:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCentral crossings: Anxiety index (fewer crossings =\\u0026nbsp;greater\\u0026nbsp;anxiety);\\u0026nbsp;Vertical activity: Exploratory behavior indicator;\\u0026nbsp;Peripheral activity: General locomotor function.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEnvironmental controls:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe samples were thoroughly cleaned\\u0026nbsp;with 75% ethanol between trials\\u0026nbsp;under consistent\\u0026nbsp;testing conditions (lighting, noise, etc.), and the experimenter\\u0026nbsp;remained outside\\u0026nbsp;the\\u0026nbsp;visual field.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.8.2 Three-\\u003c/strong\\u003e\\u003cstrong\\u003echamber social interaction test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe three-chamber test\\u0026nbsp;was used to evaluate\\u0026nbsp;social preference and novelty recognition in rodents through a standardized two-phase protocol.\\u0026nbsp;The experiment was carried out in a connected transparent three-chamber chamber,\\u0026nbsp;which is\\u0026nbsp;a classic way of assessing social skills in rats (Buffington et al., 2016).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eExperimental Protocol:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHabituation:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e7 days\\u0026nbsp;pretest: Daily 2-h\\u0026nbsp;acclimation to\\u0026nbsp;the\\u0026nbsp;test room.\\u003c/p\\u003e\\n\\u003cp\\u003eDays 1--3: 3-min center chamber exposure followed by 3-min full chamber access.\\u003c/p\\u003e\\n\\u003cp\\u003eTest day: 5-min free exploration with empty wire cages in\\u0026nbsp;the\\u0026nbsp;side chambers\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTesting Phases:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cul\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003e\\u0026nbsp;Socialization test:\\u003c/strong\\u003e\\u003c/li\\u003e\\n\\u003c/ul\\u003e\\n\\u003cp\\u003eOn\\u0026nbsp;one side of the chamber, a male Stranger 1 of the same age and from a different litter (of the same sex and age as the test animal and of the same strain that has not yet been housed in the same cage) was placed in an inverted metal coil, which was labeled\\u0026nbsp;the\\u0026nbsp;Stranger 1 cage. Only one inverted transparent metal coil was placed\\u0026nbsp;on\\u0026nbsp;the other side of the chamber, which was labeled\\u0026nbsp;the\\u0026nbsp;object cage. The test mice were placed in the empty central cage, and the activity of the test mice and the duration of mutual olfactory communication with stranger 1 or the object were observed for 10 min.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e②\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Social preference test:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAfter the first stage of\\u0026nbsp;the\\u0026nbsp;socialization test, another Stranger 2 of the same age, same sex and different\\u0026nbsp;litters\\u0026nbsp;was placed in an empty inverted metal coil and\\u0026nbsp;was\\u0026nbsp;observed for 10 min. The time spent in each side chamber and the time\\u0026nbsp;spent\\u0026nbsp;sniffing and communicating with Stranger 1 and Stranger 2 were recorded by a camera system and computer software. At the end of the experiment, the test mice and Stranger 1 and Stranger 2 inside the inverted metal coils were\\u0026nbsp;removed\\u0026nbsp;sequentially and\\u0026nbsp;returned to\\u0026nbsp;different cages. After each round of experiments, feces, urine and other excreta were promptly removed, and the experimental apparatus was wiped with 75% alcohol and dried to remove as much odor as possible from the previous animal before replacing it with the next mouse for behavioral testing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.8.3 \\u0026nbsp;Stereotyped Behavior Assessment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTwo standardized tests were employed to evaluate\\u0026nbsp;the\\u0026nbsp;repetitive\\u0026nbsp;behavior characteristics of the\\u0026nbsp;ASD models:\\u003c/p\\u003e\\n\\u003col\\u003e\\n \\u003cli\\u003e\\u003cstrong\\u003eSelf-\\u003c/strong\\u003e\\u003cstrong\\u003egrooming test\\u003c/strong\\u003e\\u003c/li\\u003e\\n\\u003c/ol\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eProtocol:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ea) 10-min habituation period\\u003c/p\\u003e\\n\\u003cp\\u003eb) 10-min observation period\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasured\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eparameters\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eCumulative time spent grooming face, limbs, body, and tail\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Marble Burying Test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe tested mice were placed in a box. The bottom of the box\\u0026nbsp;was\\u0026nbsp;lined with a layer of clean bedding approximately 5 cm thick, and the mice\\u0026nbsp;were\\u0026nbsp;placed in the box with bedding for 3 minutes to acclimatize before starting the experiment, after which the mice\\u0026nbsp;were removed\\u0026nbsp;and placed in a transit cage to wait. The bedding in the box was flattened,\\u0026nbsp;and 16 black glass balls 1.6 mm in diameter were placed in a 4 \\u0026times; 4 grid. The mice were then placed in the box and allowed to roam freely for 10 min. After 10 min, the mice were removed and photographed from all angles with a camera, and the number of beads buried was counted from the pictures and videos by three statisticians who were trained to count the number of beads from the pictures and videos and who had no knowledge of the subject: glass beads with more than 75% of the beads buried in bedding were considered buried. The enumerators had no knowledge of the grouping,\\u0026nbsp;and the final results were averaged and rounded.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.8.4 \\u0026nbsp;Morris Water Maze Behavioral Assessment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSpatial learning and memory were evaluated\\u0026nbsp;via\\u0026nbsp;the Morris water maze paradigm following established protocols from prior investigations. The experimental apparatus consisted of a round pool filled with temperature-regulated water (23\\u0026ndash;25\\u0026deg;C) rendered opaque through the addition of\\u0026nbsp;a\\u0026nbsp;food-grade titanium dioxide suspension. A circular escape platform (14 cm diameter) remained consistently positioned 1.5 cm below the water surface in the target quadrant throughout\\u0026nbsp;the\\u0026nbsp;acquisition training.\\u003c/p\\u003e\\n\\u003cp\\u003eThe training protocol comprised four daily sessions over five consecutive days, with each session containing four randomized entry-point trials.\\u0026nbsp;The subjects\\u0026nbsp;were allotted 60 s per trial to locate the submerged platform, followed by a 10 s consolidation period upon successful navigation. Animals\\u0026nbsp;that failed\\u0026nbsp;to locate the platform within the allotted time were manually assisted\\u0026nbsp;in reaching\\u0026nbsp;the platform\\u0026nbsp;via\\u0026nbsp;tail guidance. An\\u0026nbsp;intertrial\\u0026nbsp;interval of 25 min was maintained in heated recovery cages between successive trials.\\u003c/p\\u003e\\n\\u003cp\\u003eSpatial memory retention was assessed 24 h\\u0026nbsp;posttraining\\u0026nbsp;through a 60 s free-swim probe trial\\u0026nbsp;in which\\u0026nbsp;the platform\\u0026nbsp;was\\u0026nbsp;removed. All behavioral sessions were digitally captured\\u0026nbsp;via\\u0026nbsp;a video tracking system (EthoVision XT v15.0, Noldus Information Technology) for subsequent quantitative analysis.\\u0026nbsp;The primary\\u0026nbsp;outcome measures included\\u0026nbsp;the following:\\u003c/p\\u003e\\n\\u003cp\\u003eAcquisition phase: Escape latency (s) across training days.\\u003c/p\\u003e\\n\\u003cp\\u003eProbe trial:\\u0026nbsp;Platform location traversals;\\u0026nbsp;first-cross latency (s);\\u0026nbsp;swimming\\u0026nbsp;velocity (cm/s).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.9\\u003c/strong\\u003e\\u003cstrong\\u003eBiochemical analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe GSH, T-SOD, GSH-PX,\\u0026nbsp;MDA, CAT, T-NOS,\\u0026nbsp;and NO levels were measured\\u0026nbsp;via\\u0026nbsp;the corresponding biochemical kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China). The\\u0026nbsp;levels\\u0026nbsp;of IL-6, IL-10, IL-1\\u0026beta; and\\u0026nbsp;TNF-\\u0026alpha; were\\u0026nbsp;also\\u0026nbsp;measured\\u0026nbsp;via\\u0026nbsp;mouse ELISA kits (Fankel, Shanghai, China).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.10\\u003c/strong\\u003e\\u003cstrong\\u003eUltrastructural\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eanalysis via transmission electron microscopy\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;(TEM)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAt postnatal day 60 (PND 60), the mice were anesthetized via intraperitoneal injection of a ketamine‒xylazine mixture (75 mg/kg or 5 mg/kg) and subjected to transcardiac perfusion through the ascending aorta. The perfusion protocol consisted of an initial flush with ice-cold 0.01 M sodium‒potassium phosphate buffer (pH 7.4, containing 0.9% NaCl), followed by fixation with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, 20\\u0026deg;C; Sigma‒Aldrich, St. Louis, MO, USA). Tissue blocks (approximately 1 mm\\u0026sup3;) were dissected from the frontal cerebral cortex and hippocampal CA1 region across all experimental and control groups for ultrastructural examination.\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing 20 hours of primary fixation in ice-cold fixative, the samples were postfixed in a solution containing 1% osmium tetroxide (OsO₄) and 0.8% potassium ferrocyanide [K₄Fe(CN)₆]. After dehydration through a graded ethanol series, the tissue blocks were embedded in epoxy resin (Epon 812). Ultrathin sections (60 nm thickness) were prepared via an ultramicrotome, double-stained with uranyl acetate and lead citrate, and examined via transmission electron microscopy (JEM-1200EX, Jeol, Japan). Digital images were acquired via a MORADA CCD camera coupled with iTEM 1233 imaging software (Olympus, Japan). The data are presented as the means \\u0026plusmn; SEMs\\u0026nbsp;from 3 independent animals per group (VPA-exposed vs. control).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1.11\\u003c/strong\\u003e\\u003cstrong\\u003eStatistical\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eanalysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eQuantitative\\u0026nbsp;\\u003c/strong\\u003edata expression: All\\u0026nbsp;continuous variables\\u0026nbsp;are\\u0026nbsp;expressed as\\u0026nbsp;the\\u0026nbsp;mean \\u0026plusmn; standard deviation (x̅\\u0026plusmn;s).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e1. Normality and Homogeneity of Variance Assessment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eData normality was evaluated\\u0026nbsp;via\\u0026nbsp;the\\u0026nbsp;Shapiro‒Wilk\\u0026nbsp;test prior to statistical analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2. Comparative Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.1 Two-Group Comparisons\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eParametric\\u0026nbsp;conditions:\\u0026nbsp;When\\u0026nbsp;the\\u0026nbsp;data followed a normal distribution and homogeneity of variance was confirmed by Levene\\u0026apos;s test (P\\u0026gt;0.05), an\\u0026nbsp;independent two-sample t\\u0026nbsp;test was applied.\\u003c/p\\u003e\\n\\u003cp\\u003eNonparametric conditions: For nonnormally\\u0026nbsp;distributed data or unequal variances,\\u0026nbsp;the Mann‒Whitney\\u0026nbsp;U test (Kruskal‒Wallis) was employed.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.2 Categorical Variable Comparisons\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePearson\\u0026rsquo;s \\u0026chi;\\u0026sup2; test was used when all expected frequencies\\u0026nbsp;were\\u0026nbsp;\\u0026ge; 5.\\u003c/p\\u003e\\n\\u003cp\\u003eFisher\\u0026rsquo;s exact test was adopted if any expected frequency\\u0026nbsp;was\\u0026nbsp;\\u0026lt; 5.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.3\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eMultigroup\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;Comparisons\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eParametric data:\\u003c/strong\\u003eOne-way analysis of variance (ANOVA) with Tukey\\u0026rsquo;s HSD post hoc test was performed.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNonparametric\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;data:\\u003c/strong\\u003e The Kruskal‒Wallis test followed by Dunn\\u0026rsquo;s multiple comparison correction was used.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e2.1\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eDose- and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003etime-dependent maternal toxicity of VPA\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePrenatal sodium valproate (VPA) administration\\u0026nbsp;has\\u0026nbsp;dose- and gestational timing-dependent\\u0026nbsp;effects\\u0026nbsp;on maternal survival and pregnancy maintenance.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;(1) Toxic reaction of pregnant mice (Table 1): After a single intraperitoneal injection of VPA (600 mg/kg) in the traditional model group, pregnant C57BL/6 mice presented acute toxicity symptoms at 3\\u0026ndash;5 min, which included stiffness of the limbs, motor disorders, paralysis with the eyes closed, and respiratory and circulatory disorders, which ultimately led to death. In contrast, the symptoms of the fractionally administered groups were significantly relieved: the time to the onset of symptoms of rigidity was significantly delayed in modified group 1 (13\\u0026ndash;15 min) and modified group 2 (8\\u0026ndash;10 min), and they maintained basic motor ability during the 30-min observation period.\\u003c/p\\u003e\\n\\u003cp\\u003e(2) Survival analysis\\u0026nbsp;(Table 1):\\u0026nbsp;Within\\u0026nbsp;6 h after drug administration, the mortality rate of the traditional group\\u0026nbsp;reached\\u0026nbsp;25% (6/24), whereas there were no\\u0026nbsp;deaths\\u0026nbsp;in modified groups 1 and 2 (0/24), and the difference was statistically significant (p\\u0026lt;0.05) (Fisher\\u0026apos;s exact test).\\u003c/p\\u003e\\n\\u003cp\\u003e(3) Pregnancy outcome\\u0026nbsp;(Table 1): the abortion rate was 54.17% (13/24) in the traditional group and significantly reduced to 29.17% (7/24; p\\u0026lt;0.05) (Fisher\\u0026apos;s exact test)\\u0026nbsp;in modified group 1. Notably, the miscarriage rate in modified group 2 further decreased to 12.5% (3/24), which\\u0026nbsp;was\\u0026nbsp;highly\\u0026nbsp;significantly different from that in\\u0026nbsp;the traditional group (Fisher\\u0026apos;s exact test, p\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 1. Comparison of production in pregnant C57BL/6 mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"100%\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 20px;\\\"\\u003e\\n \\u003cp\\u003eGroup\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 30px;\\\"\\u003e\\n \\u003cp\\u003eAbortion (n, %)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 25px;\\\"\\u003e\\n \\u003cp\\u003eMortality(n, %)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 23px;\\\"\\u003e\\n \\u003cp\\u003eNormal (n, %)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 20px;\\\"\\u003e\\n \\u003cp\\u003eControl\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 30px;\\\"\\u003e\\n \\u003cp\\u003e0 (0%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 25px;\\\"\\u003e\\n \\u003cp\\u003e0 (0%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 23px;\\\"\\u003e\\n \\u003cp\\u003e24 (100%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 20px;\\\"\\u003e\\n \\u003cp\\u003eTraditional\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 30px;\\\"\\u003e\\n \\u003cp\\u003e13（54.17%）\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 25px;\\\"\\u003e\\n \\u003cp\\u003e6 (25%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 23px;\\\"\\u003e\\n \\u003cp\\u003e5 (20.83%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 20px;\\\"\\u003e\\n \\u003cp\\u003eModified 1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 30px;\\\"\\u003e\\n \\u003cp\\u003e7 (29.17%)*\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 25px;\\\"\\u003e\\n \\u003cp\\u003e0 (0%)*\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 23px;\\\"\\u003e\\n \\u003cp\\u003e17 (70.83%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 20px;\\\"\\u003e\\n \\u003cp\\u003eModified 2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 30px;\\\"\\u003e\\n \\u003cp\\u003e3 (12.5%)**\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 25px;\\\"\\u003e\\n \\u003cp\\u003e0 (0%)*\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 23px;\\\"\\u003e\\n \\u003cp\\u003e21(87.5%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.2 Dose- and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003etime-dependent teratogenic effects of prenatal VPA exposure\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll newborn mice in the control group presented normal developmental phenotypes, including reddish skin color, complete body shape and no malformations visible to the naked eye (n=106). In contrast, the offspring of the traditional model group presented significant developmental abnormalities: 15.63% (5/32) of the littermates presented typical deformities, such as shortened limbs, underdeveloped eyes (including one case of monocular deformity), and missing auricles. Statistical analysis revealed that the malformation rate of this group was significantly greater than that of the control group, confirming that a single high-dose intraperitoneal injection of VPA (600 mg/kg) induced severe embryotoxicity (P\\u0026lt;0.05) (Fisher\\u0026apos;s exact test) (Table 2). Notably, the teratogenic effect due to embryotoxicity was significantly reduced in the modified dosing regimens (Group 1 and Group 2), and the offspring（Figure1A）malformation rate was 0% in both groups, which was not significantly different from that of the control group (P \\u0026gt;0.05). The C57BL/6 mouse model of ASD, generated through an optimized experimental protocol, exhibited a classical tail curvature phenotype（Figure1C）in the majority of subjects. This characteristic morphological feature served as a primary phenotypic marker for successful model validation.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.3 Dose- and Time-Dependent Developmental Impairments in Male Littermates by VPA Exposure\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;(1) 4-day survival rate (Table 2): The 4-day survival rate of C57BL/6 male littermates in the traditional model group was 78.13% (25/32), which was significantly lower than that in modified group 1 (100%, 76/76) (P\\u0026lt;0.001) (Fisher\\u0026apos;s exact test) and modified group 2 (100%, 87/87) (P\\u0026lt;0.001) (Fisher\\u0026apos;s exact test), suggesting that the optimization of the VPA exposure regimen could effectively reduce the early mortality of neonatal mice. (2) Inhibition of body weight development (Figure 2A): male littermates in all intervention groups (traditional, modified group 1 and modified group 2) presented significant body weight inhibition beginning 21 days after birth (P\\u0026lt;0.01). (3) Lagging tail length development (Figure 2B): Tail lengths (Figure 1B) of male littermates across experimental groups were measured. the tail lengths of male littermates in all intervention groups (traditional, modified group 1 and modified group 2) were significantly shorter than those of the control group beginning at 28 d after birth (P\\u0026lt;0.01). (4) Tail curvature rate (see Table 2): The incidence of bent tails in the traditional group was 40% (10/25), which was significantly lower than that in modified group 1 (82.9%, 63/76) (P\\u0026lt;0.001) (Fisher\\u0026apos;s exact test) and modified group 2 (88.51%, 77/87) (P\\u0026lt;0.001) (Fisher\\u0026apos;s exact test). Curved tail deformities in rodents are thought to be associated with minor neural tube closure defects. (5) Age of positive incisor eruption (Figure 2C): The age of positive incisor eruption in C57BL/6 pregnant mice in modified group 1 and modified group 2 was significantly delayed compared with that in the control group (P\\u0026lt;0.01), but there was no significant difference between the traditional model group and the control group (P\\u0026gt;0.05). (6) Age of positive fur development (Figure 2D): Male C57BL/6 model mice in the traditional model group and modified group 1 exhibited positive abdominal hair growth, which was significantly delayed compared with that of the control group (P\\u0026lt;0.05). Compared with the control group, modified group 2 was significantly delayed (P\\u0026lt;0.01). These findings suggest that the injection of VPA during pregnancy can significantly delay the development of incisor eruption, eye opening and fur development in male C57BL/6 model mice in the modified model group. (7) Age of positive eye opening (Figure 2E): Compared with that in the control group, the age of positive eye opening in the modified group 2 was significantly delayed (P\\u0026lt;0.01), the age of positive eye opening in the modified group 1 was significantly delayed (P\\u0026lt;0.01), and there was no significant difference between the traditional model group and the control group (P\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 2\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e4-day survival rate\\u003c/strong\\u003e\\u003cstrong\\u003e,\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003etail curvature rate\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eincidence of\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;deformities\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ein newborns\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e(n,%)\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGroup\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e4-day survival rate (n, %)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTail curvature rate (n, %)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eappearance deformities rate (n, %)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTotality\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eControl\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e106 (100%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e0 (0%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e0 (0%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e106\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eTraditional\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e25（78.13%）\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e10 (40%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e5 (15.63%)*\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e32\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eModified 1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e76(100%)***\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e63(82.90%)****\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0 (0%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e76\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eModified 2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e87(100%)***\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e77 (88.51%)****\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e0 (0%)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003e87\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.4\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eDose-\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eand\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003etime-dependent effects\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;of VPA on\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eneurological development\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe experimental results demonstrated that variations in valproic acid (VPA) exposure\\u0026nbsp;dose\\u0026nbsp;and timing significantly impacted neurodevelopmental behavioral indices in male C57BL/6 offspring (Figure 3).\\u0026nbsp;The detailed\\u0026nbsp;analyses are as follows:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Delayed Reflex Development\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSurface righting reflex\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e(Figure 3D): The number of positive reflex days in the modified group 2 and modified group 1 was significantly lower than that in the control group (P\\u0026lt;0.01), whereas no significant difference was observed between the traditional model group (P\\u0026gt;0.05). \\u003cstrong\\u003eCliff avoidance reflex\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e(Figure 3A): Compared with the control group, both Modified Groups 1 and 2 presented delayed positive reflex days (P\\u0026lt;0.05), with no significant difference between the traditional model group and the control group (P\\u0026gt;0.05). \\u003cstrong\\u003eAir righting reflex\\u003c/strong\\u003e (Figure 3C): Significant delays in positive reflex days were observed in Modified Group 1 and the traditional model group (P\\u0026lt;0.05), whereas Modified Group 2 showed a more pronounced delay (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Altered Motor Coordination\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCrawling behavior\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e(Figure 3F): The\\u0026nbsp;number of\\u0026nbsp;positive reflex days in\\u0026nbsp;the modified group\\u0026nbsp;2 and the traditional model group\\u0026nbsp;was\\u0026nbsp;significantly\\u0026nbsp;lower than that in the control group\\u0026nbsp;(P\\u0026lt;0.05), with\\u0026nbsp;modified group\\u0026nbsp;1 displaying a\\u0026nbsp;greater\\u0026nbsp;delay (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIn the bar\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;holding test\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e(Figure 3H),\\u0026nbsp;Modified Group 2\\u0026nbsp;presented a\\u0026nbsp;significant lag in positive reflex days (P\\u0026lt;0.01), followed by Modified Group 1 (P\\u0026lt;0.05), whereas the traditional model group\\u0026nbsp;presented\\u0026nbsp;no difference from\\u0026nbsp;the control group\\u0026nbsp;(P\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(3) Abnormal\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003esensory\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003estress responses\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuditory startle reflex\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e(Figure 3E): The reflex latency in Modified Group 2 was significantly shorter than that in the control group (P\\u0026lt;0.001), with a similar trend in Modified Group 1 (P\\u0026lt;0.05), suggesting that midgestational VPA exposure may increase auditory sensitivity in offspring. \\u003cstrong\\u003eNegative geotaxis reflex\\u0026nbsp;\\u003c/strong\\u003e(Figure 3B): Modified Groups 1 and 2 presented significant delays in positive reflex days (P\\u0026lt;0.01), whereas the traditional model group remained unaffected (P\\u0026gt;0.05). Tail-flick photic test reflex (Figure 3G): Latency was significantly prolonged in Modified Group 1 and the traditional model group (P\\u0026lt;0.05), with an even greater prolongation in Modified Group 2 (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.5 Open Field Test Behavioral Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe open field test is designed to assess autonomous exploratory behaviors and anxiety-like behavioral traits in experimental animals within a novel environment.\\u003c/p\\u003e\\n\\u003cp\\u003eThe experimental\\u0026nbsp;results revealed that valproic acid (VPA) exposure significantly suppressed exploratory behaviors and enhanced anxiety-like responses in C57BL/6 mice (Figure 4).\\u0026nbsp;The detailed\\u0026nbsp;findings are as follows:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.5.1 Exploratory\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ebehavior\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCross center grid\\u0026nbsp;\\u003c/strong\\u003e(Figure 4A): The number of crossings in Modified Group 1 and the traditional model group was significantly lower than that in the control group (P\\u0026lt;0.05), whereas Modified Group 2 exhibited a more pronounced reduction (P\\u0026lt;0.01). \\u003cstrong\\u003eInner area distance\\u003c/strong\\u003e (Figure 4B): The movement distances in Modified Groups 1 and 2, as well as those in the traditional model group, were significantly lower than those in the control group (P\\u0026lt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.5.2 Enhanced Anxiety-like Behaviors\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eInner area time\\u003c/strong\\u003e (Figure 4C): The activity time in Modified Group 1 and the traditional model group was significantly shorter than that in the control group (P\\u0026lt;0.05), with Modified Group 2 showing an even greater reduction (P\\u0026lt;0.01). \\u003cstrong\\u003eVertical score\\u003c/strong\\u003e (Figure 4D): Scores in Modified Group 1 significantly decreased (P\\u0026lt;0.05), whereas those in Modified Group 2 substantially decreased (P\\u0026lt;0.001). No significant difference was observed between the traditional model group and the control group (P\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.6 Three-\\u003c/strong\\u003e\\u003cstrong\\u003echamber social behavioral analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe three-chamber social test was utilized to evaluate social motivation (0\\u0026ndash;10\\u0026nbsp;min) and social novelty preference (10\\u0026ndash;20\\u0026nbsp;min) in experimental animals. By measuring exploration time toward unfamiliar conspecifics (Stranger 1/2) versus empty cages/objects, this assay quantified social competence and cognitive flexibility\\u0026nbsp;(Figure 5).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eI. Social Motivation Phase (Socialability,\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e0\\u0026ndash;10\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;min)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe experimental\\u0026nbsp;results demonstrated that valproic acid (VPA) exposure significantly impaired social interaction in C57BL/6 mice (Figure 5A):\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Spatial exploration preference\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStranger 1 cage duration:\\u0026nbsp;Compared with the control group, the traditional model group, modified group 1, and modified group 2 groups presented significantly shorter durations in the Stranger 1 cage\\u0026nbsp;(P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003eEmpty cage duration: These experimental groups\\u0026nbsp;presented\\u0026nbsp;a marked increase in time spent in the empty cage (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Social interaction behavior\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStranger 1 sniffing time: All VPA-exposed groups\\u0026nbsp;presented\\u0026nbsp;significantly\\u0026nbsp;shorter\\u0026nbsp;interaction\\u0026nbsp;times\\u0026nbsp;with Stranger 1\\u0026nbsp;than did the\\u0026nbsp;controls (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003eObject sniffing time: Exploration time toward objects also notably decreased in\\u0026nbsp;the\\u0026nbsp;experimental groups (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003eConclusion: VPA exposure induced\\u0026nbsp;the\\u0026nbsp;avoidance of social stimuli (Stranger 1) and\\u0026nbsp;a\\u0026nbsp;preference for nonsocial environments (empty cages), indicating severe impairment of social motivation (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eII. Social\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003enovelty preference phase (10-\\u003c/strong\\u003e\\u003cstrong\\u003e-20 min)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing the introduction of a novel social stimulus (Stranger 2),\\u0026nbsp;the\\u0026nbsp;behavioral patterns were as follows (Figure 5B):\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Spatial exploration patterns\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStranger 1 cage duration:\\u0026nbsp;The experimental\\u0026nbsp;groups spent significantly more time in the Stranger 1 cage than\\u0026nbsp;the control groups did\\u0026nbsp;(P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003eStranger 2 cage duration: Time in the Stranger 2 cage was dramatically reduced in\\u0026nbsp;the\\u0026nbsp;experimental groups (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Social interaction disparities\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStranger 1 sniffing time:\\u0026nbsp;The interaction\\u0026nbsp;time with Stranger 1 increased in\\u0026nbsp;the\\u0026nbsp;experimental groups (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003eStranger 2 sniffing time: Exploration of the novel social stimulus (Stranger 2) was significantly diminished (P\\u0026lt;0.0001).\\u003c/p\\u003e\\n\\u003cp\\u003eConclusion: VPA-exposed mice lacked typical novelty-seeking behavior, suggesting a restricted interest range and compromised social cognitive flexibility..\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.7\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eAnalysis of\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003erepetitive stereotypic behavior\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Buried marble\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;test\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe marble-burying test is a classical paradigm for assessing repetitive stereotypic behaviors in rodents.\\u0026nbsp;The experimental\\u0026nbsp;results demonstrated that VPA exposure significantly increased marble-burying behavior in C57BL/6 mice (Figure 4E).\\u003c/p\\u003e\\n\\u003cp\\u003eCompared with the control group,\\u0026nbsp;Modified Group 2\\u0026nbsp;presented\\u0026nbsp;a marked increase in\\u0026nbsp;the\\u0026nbsp;buried marble count (P\\u0026lt;0.01), followed by Modified Group 1 (P\\u0026lt;0.05). No significant difference was observed in the\\u0026nbsp;traditional model group\\u0026nbsp;(P\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Self\\u003c/strong\\u003e\\u003cstrong\\u003e-grooming\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eVPA-exposed mice displayed excessive self-grooming behavior (Figure 4F):\\u003c/p\\u003e\\n\\u003cp\\u003eCompared with the control group, the traditional model group, modified Group 1, and modified Group 2 all presented significantly greater grooming frequencies\\u0026nbsp;(P\\u0026lt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003eConclusion: C57BL/6 mice\\u0026nbsp;presented\\u0026nbsp;a dose-dependent increase in repetitive stereotypic behaviors following VPA exposure, suggesting potential neurodevelopmental anomalies linked to dysfunction in the basal ganglia-cortical circuit.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.8 Analysis of Neuroinflammatory Levels in the Prefrontal Cortex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eELISAs\\u0026nbsp;revealed that valproic acid (VPA) exposure significantly altered the inflammatory cytokine profile in the prefrontal cortex of C57BL/6 mice (\\u003cstrong\\u003eFigure 6\\u003c/strong\\u003e).\\u0026nbsp;The key\\u0026nbsp;findings are as follows:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Upregulation of proinflammatory cytokines\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIL-1\\u0026beta;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e(Figure 6A):\\u0026nbsp;\\u003c/strong\\u003eThe IL-1\\u0026beta; levels in the Traditional Model Group,\\u0026nbsp;Modified Group 1 and Modified Group 2 were significantly\\u0026nbsp;greater\\u0026nbsp;than\\u0026nbsp;those\\u0026nbsp;in the Control\\u0026nbsp;Group\\u0026nbsp;(P\\u0026lt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIL-6\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e(Figure 6B):\\u0026nbsp;\\u003c/strong\\u003eIL-6 levels in the\\u0026nbsp;traditional model group and modified group\\u0026nbsp;1 were markedly elevated compared\\u0026nbsp;with those in the control group\\u0026nbsp;(P\\u0026lt;0.05), with\\u0026nbsp;modified group\\u0026nbsp;2 showing further exacerbation (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003eThe \\u003cstrong\\u003eTNF-\\u0026alpha;\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;content\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e(Figure 6C)\\u003c/strong\\u003e increased significantly in the traditional model group and modified group 1 (P\\u0026lt;0.05), and a similar trend was observed in modified group 2 (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(2) Downregulation of anti\\u003c/strong\\u003e\\u003cstrong\\u003e-inflammatory\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ecytokines\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIL-10\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e(Figure 6D):\\u0026nbsp;\\u003c/strong\\u003eIL-10 levels in the\\u0026nbsp;traditional model group and modified group\\u0026nbsp;2 were significantly\\u0026nbsp;lower than those in the control group\\u0026nbsp;(P\\u0026lt;0.05), with\\u0026nbsp;modified group\\u0026nbsp;1 demonstrating an even greater reduction (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003eConclusion: VPA exposure disrupted the proinflammatory/anti-inflammatory balance in the prefrontal cortex of C57BL/6 mice. Modified Group 2 exhibited the most significant enhancement of inflammatory responses, suggesting that its intervention strategy may exacerbate neuroinflammatory processes.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.9\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eAnalysis of Oxidative Stress Levels in the Prefrontal Cortex\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eELISAs\\u0026nbsp;demonstrated that valproic acid (VPA) exposure significantly disrupted oxidative stress homeostasis in the prefrontal cortex of C57BL/6 mice (Figure 6).\\u0026nbsp;The specific\\u0026nbsp;findings are as follows:\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(1) Suppression\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;of Antioxidant Systems\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCatalase (CAT) (Figure 6E):\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eCAT levels in the\\u0026nbsp;traditional model group and modified group\\u0026nbsp;1 were significantly\\u0026nbsp;lower than those in the control\\u0026nbsp;group (P\\u0026lt;0.05), with\\u0026nbsp;modified group\\u0026nbsp;2 exhibiting a more pronounced decrease (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGlutathione\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eperoxidase\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;(GSH-Px)\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eactivity\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e(Figure 6F):\\u0026nbsp;\\u003c/strong\\u003eGSH-Px activity\\u0026nbsp;decreased\\u0026nbsp;significantly in the\\u0026nbsp;traditional model group and modified group\\u0026nbsp;1 (P\\u0026lt;0.05),\\u0026nbsp;whereas that in modified group 2 further decreased\\u0026nbsp;(P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGlutathione (GSH) (Figure 6G):\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eThe\\u0026nbsp;\\u003c/strong\\u003eGSH content decreased significantly in the\\u0026nbsp;traditional model group and modified group\\u0026nbsp;1 (P\\u0026lt;0.05), with a more marked reduction in\\u0026nbsp;the modified group\\u0026nbsp;2 (P\\u0026lt;0.01).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSuperoxide Dismutase (SOD) (Figure 6K):\\u0026nbsp;\\u003c/strong\\u003eSOD levels were significantly lower in Modified Group 1 (P\\u0026lt;0.05) and Modified Group 2 (P\\u0026lt;0.01), whereas no significant change was observed in the Traditional Model Group (P\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e(2) Elevation\\u0026nbsp;of Oxidative Damage Markers\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe malondialdehyde\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;(MDA)\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003econtent\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003e(Figure 6H)\\u0026nbsp;\\u003c/strong\\u003ewas\\u0026nbsp;significantly\\u0026nbsp;greater\\u0026nbsp;in all\\u0026nbsp;the\\u0026nbsp;experimental groups (traditional model group, modified group 1, and modified group 2) than in the control group\\u0026nbsp;(P\\u0026lt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNitric\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eoxide synthase\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;(NOS) (Figure 6I) and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003enitric oxide\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;(NO) (Figure 6J):\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eNOS activity and NO levels were significantly elevated in the\\u0026nbsp;traditional model group\\u0026nbsp;(P\\u0026lt;0.05) and\\u0026nbsp;modified group\\u0026nbsp;2 (P\\u0026lt;0.01),\\u0026nbsp;whereas modified group\\u0026nbsp;1 showed no significant changes (P\\u0026gt;0.05).\\u003c/p\\u003e\\n\\u003cp\\u003eConclusion: VPA exposure induced comprehensive impairment of antioxidant capacity and significant accumulation of oxidative damage markers in the prefrontal cortex of C57BL/6 mice.\\u0026nbsp;The modified\\u0026nbsp;Group 2\\u0026nbsp;group presented\\u0026nbsp;the most severe\\u0026nbsp;degree of\\u0026nbsp;oxidative stress dysregulation, suggesting that VPA exacerbates redox imbalance in a dose-dependent manner.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.10 Morris\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ewater maze behavioral analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.10.1 Assessment of Learning and Memory Capacity\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe Morris water maze (MWM)\\u0026nbsp;is\\u0026nbsp;a cognitively demanding task for rodents\\u0026nbsp;that involves\\u0026nbsp;complex memory processes. The protocol comprises two phases: spatial acquisition trials and spatial probe trials.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSpatial Acquisition Trials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDuring training,\\u0026nbsp;the\\u0026nbsp;VPA-induced ASD model mice exhibited undirected, random search patterns to locate the hidden platform (Figure 7F). In contrast, control mice demonstrated goal-oriented navigation strategies, with some individuals swimming directly to the platform on\\u0026nbsp;the basis of\\u0026nbsp;spatial memory (Figure 7G). Compared\\u0026nbsp;with control mice, VPA-induced ASD mice\\u0026nbsp;presented\\u0026nbsp;significantly prolonged escape latencies (P \\u0026lt; 0.01; Figure 7C).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSpatial Probe Trials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eQuadrant Residence Time:\\u0026nbsp;\\u003c/strong\\u003eVPA-induced ASD mice showed no quadrant preference (P \\u0026gt; 0.05), whereas control mice spent significantly more time in the target quadrant than in\\u0026nbsp;the other quadrants did\\u0026nbsp;(P \\u0026lt; 0.0001; Figure 7E). Swimming trajectories revealed that control mice predominantly navigated the original platform quadrant, repeatedly crossing the target area (Figure 7I),\\u0026nbsp;whereas\\u0026nbsp;ASD model mice engaged in aimless exploration across all quadrants (Figure 7H).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePlatform\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ecrossings\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eThe number of platform crossings was significantly\\u0026nbsp;lower\\u0026nbsp;in VPA-induced ASD mice\\u0026nbsp;than in control mice\\u0026nbsp;(P \\u0026lt; 0.0001; Figure 7A).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTarget\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003equadrant duration\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eASD model mice spent markedly less time in the target quadrant than\\u0026nbsp;control mice did\\u0026nbsp;(P \\u0026lt; 0.0001; Figure 7D), indicating impaired spatial recognition and memory retention.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSwimming\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003edistance\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u003c/strong\\u003e No significant intergroup differences in total swimming distance were observed (P \\u0026gt; 0.05; Figure 7B), confirming preserved motor function in ASD model mice.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eVPA-induced ASD mice exhibited pronounced deficits in spatial learning and memory, as evidenced by prolonged escape latencies, random search strategies, and reduced target quadrant preference. These impairments occurred independently of locomotor dysfunction, underscoring the specificity of cognitive deficits in the ASD model.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.11 TEM Analysis Reveals Synaptic Ultrastructural Pathologies in VPA-induced ASD Mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eUltrastructural\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003efeatures\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;of\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ethe control group\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTransmission electron microscopy (TEM) analysis\\u0026nbsp;revealed\\u0026nbsp;characteristic ultrastructural features in the prefrontal cortex (PFC) and hippocampal CA1 region neurons of control mice (Figure 8).\\u0026nbsp;The synaptic\\u0026nbsp;clefts exhibited narrow spacing, with sharply defined and densely stained postsynaptic densities (PSDs). Synaptic vesicles (SVs) were densely clustered in presynaptic terminals, predominantly adjacent to the presynaptic membrane\\u0026nbsp;(Figure 8C).\\u0026nbsp;The mitochondria\\u0026nbsp;displayed well-organized\\u0026nbsp;crista\\u0026nbsp;structures with intact membranes\\u0026nbsp;and\\u0026nbsp;no signs of swelling or shrinkage\\u0026nbsp;(Figure 8A).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePathological\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ealterations\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;in\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ethe\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eVPA-induced ASD\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003emodel group (modified group\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;2)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCompared with the control ASD model group, the\\u0026nbsp;VPA-exposed ASD model group\\u0026nbsp;presented\\u0026nbsp;significant synaptic ultrastructural abnormalities (Figure 8D). Presynaptic terminals exhibited marked SV depletion, with\\u0026nbsp;the\\u0026nbsp;complete absence of SVs in localized areas. Synaptic membrane disorganization\\u0026nbsp;manifests\\u0026nbsp;as blurred and thickened synaptic clefts, accompanied by indistinct boundaries between pre- and postsynaptic membranes. Notably, abnormal PSD thickening with reduced electron density was observed in select synapses.\\u0026nbsp;The mitochondrial\\u0026nbsp;pathologies included\\u0026nbsp;crista\\u0026nbsp;disorganization and membrane rupture, presenting as alternating swelling and shrinkage\\u0026nbsp;(Figure 8B).\\u003c/p\\u003e\\n\\u003cp\\u003eThese ultrastructural perturbations indicate impaired SV release at presynaptic terminals and disrupted postsynaptic signaling, potentially altering the excitatory-inhibitory balance. Mitochondrial structural derangements likely exacerbate metabolic insufficiency.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eAutism spectrum disorder (ASD) has emerged as a significant global health concern, with its\\u0026nbsp;increasing\\u0026nbsp;prevalence imposing a heavy burden on families and society[21]. As reported by the US CDC, the prevalence of ASD has reached 1.7%, and in China, it is already 1%[22].\\u0026nbsp;There\\u0026nbsp;are nearly 80 million patients\\u0026nbsp;worldwide, and\\u0026nbsp;this number\\u0026nbsp;has surpassed the incidence of childhood tumors, diabetes, leukemia, and AIDS combined[23]. The high cost of caring for\\u0026nbsp;approximately\\u0026nbsp;one-third of ASD children throughout their lives places substantial economic strain[24]. For\\u0026nbsp;example, the annual social cost of ASD in the United States is approximately $236 billion. Given its impact, ASD has become a major public health issue and the leading cause of childhood disability, even being referred to as “mental cancer” with no current cure, sparking extensive research efforts to understand its etiology, as reflected in Science's list of challenging scientific questions.\\u003c/p\\u003e\\n\\u003cp\\u003ePrevious studies have also emphasized the importance of the timing and dose of VPA exposure during pregnancy. Servadio et al. (2018b)\\u0026nbsp;reported\\u0026nbsp;that the most vulnerable period for VPA-induced pathogenesis in rodents is around embryonic day 12.5, which is a critical period for neural tube closure[15]. Kim et al. (2011) compared VPA exposure at different gestational days in rats and confirmed that exposure\\u0026nbsp;at approximately\\u0026nbsp;day 12 of pregnancy is most likely to cause offspring morbidity[25]. Williams et al suggested that in humans, exposure to VPA,\\u0026nbsp;such as\\u0026nbsp;teratogenic chemicals,\\u0026nbsp;between the\\u0026nbsp;20th and\\u0026nbsp;24th\\u0026nbsp;days\\u0026nbsp;of pregnancy, which is equivalent to the\\u0026nbsp;9th and\\u0026nbsp;12th\\u0026nbsp;days\\u0026nbsp;of pregnancy, is associated with a high incidence of ASD[26]. Most studies use a VPA dose range of\\u0026nbsp;300–800\\u0026nbsp;mg/kg, with 600 mg/kg being a common dose. However, our\\u0026nbsp;previous study revealed\\u0026nbsp;that a single injection of 600 mg/kg VPA in rats and mice led to high mortality and abortion rates and a low modeling success rate,\\u0026nbsp;which is\\u0026nbsp;consistent with the findings of other scholars[27].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;In this study, we aimed to optimize the establishment of an ASD model in\\u0026nbsp;C57BL/6J\\u0026nbsp;mice\\u0026nbsp;via the use of\\u0026nbsp;valproic acid (VPA), addressing the limitations of traditional methods. Our preexperiment,\\u0026nbsp;which followed\\u0026nbsp;the conventional approach of a single intraperitoneal injection of 600 mg/kg VPA, led to high mortality and abortion rates in pregnant mice, resulting in a low modeling success rate and\\u0026nbsp;a\\u0026nbsp;significant waste of resources. This\\u0026nbsp;finding\\u0026nbsp;is consistent with the understanding that high-dose VPA has strong toxic side effects.\\u003c/p\\u003e\\n\\u003cp\\u003eThe innovative aspect of our study lies in the administration method. Conventionally, mice and rats are mated at 17:00, and pregnancy is checked\\u0026nbsp;at approximately\\u0026nbsp;9:00 the next morning, with a 16-hour interval during which the actual fertilization time varies greatly among individuals.\\u0026nbsp;The traditional\\u0026nbsp;literature typically calculates the fertilization time as\\u0026nbsp;approximately\\u0026nbsp;2:00 am for determining the 12.5-day dosing time, but this is inaccurate. Single-dose administration can ensure accurate dosing for\\u0026nbsp;only approximately\\u0026nbsp;1/3 of the animals, and two-dose administration can achieve\\u0026nbsp;only\\u0026nbsp;2/3 accuracy. In contrast, our novel three-dose administration strategy addresses this issue. By administering three doses\\u0026nbsp;of VPA\\u0026nbsp;at different times around E12.5 (for example, in C57BL/6 mice, the modified model 1 group received doses of\\u0026nbsp;200 mg/kg on E12,\\u0026nbsp;300 mg/kg on E12.5, and\\u0026nbsp;200 mg/kg on E13; the modified model 2 group received\\u0026nbsp;300 mg/kg on E11.5,\\u0026nbsp;400 mg/kg on E12.5, and\\u0026nbsp;300 mg/kg on E13.5), we can ensure that all\\u0026nbsp;the\\u0026nbsp;animals receive the drug at the most accurate time, regardless of whether they conceive earlier or later. This significantly improves the modeling efficiency, reduces experimental costs, minimizes side effects,\\u0026nbsp;and aligns\\u0026nbsp;better with animal welfare principles.\\u003c/p\\u003e\\n\\u003cp\\u003eTo further\\u0026nbsp;increase\\u0026nbsp;the precision of the dosing time, several additional strategies could be considered. One approach could be to use more advanced detection techniques to determine the exact time of fertilization. For example,\\u0026nbsp;noninvasive\\u0026nbsp;imaging methods or hormonal assays could be employed to detect early signs of fertilization more accurately. By closely monitoring hormonal changes in female animals, such as the levels of progesterone or luteinizing hormone, it may be possible to pinpoint the time of ovulation and subsequent fertilization more precisely. This would allow for a more individualized dosing schedule tailored to the specific fertilization time of each animal.\\u003c/p\\u003e\\n\\u003cp\\u003eOur modified method not only reduces the side effects on pregnant mice but also better mimics the clinical situation of pregnant women taking VPA-related drugs multiple times. The traditional single-injection method caused severe acute toxicity in pregnant C57BL/6 mice, with a 16.7% mortality rate within 6 h and a 50% abortion rate. In contrast, the modified groups had no mortality, and their abortion rates were significantly\\u0026nbsp;lower. This is because multidose administration allows the body to adapt to the drug gradually, and the drug remains in the body for a longer and more stable period, which may cause more severe damage to the brain development of offspring in a more physiological way.\\u003c/p\\u003e\\n\\u003cp\\u003eThe offspring of\\u0026nbsp;the\\u0026nbsp;mice in the modified groups also exhibited more prominent ASD-related behaviors. In behavioral tests such as the open-field test, three-chamber social test, and self-grooming test, the male offspring of C57BL/6 mice\\u0026nbsp;presented\\u0026nbsp;characteristics similar to those of ASD patients, such as reduced exploration, social deficits, and increased repetitive behaviors.\\u003c/p\\u003e\\n\\u003cp\\u003eCompared with other\\u0026nbsp;models, our improved ASD model\\u0026nbsp;results in\\u0026nbsp;a significantly\\u0026nbsp;greater\\u0026nbsp;delivery rate\\u0026nbsp;to\\u0026nbsp;pregnant mice and a\\u0026nbsp;greater\\u0026nbsp;rate of external malformations in newborn pups.\\u0026nbsp;These findings indicate\\u0026nbsp;that our method can more effectively simulate the core symptoms of ASD, greatly improving the modeling efficiency, reducing experimental costs, and protecting animal welfare. In conclusion, our optimized modeling method provides a more reliable and efficient tool for studying the pathogenesis of ASD and developing potential therapeutic strategies, which may contribute to the ultimate conquest of this disorder.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSummary:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eOptimized Maternal Safety:\\u0026nbsp;\\u003c/strong\\u003eThe phased dosing regimen significantly improved maternal survival rates and reduced abortion rates, resolving acute toxicity challenges\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003cstrong\\u003eRobust ASD-like\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ephenotypes\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u003c/strong\\u003e Offspring\\u0026nbsp;exhibit\\u0026nbsp;core behavioral deficits, including impaired social interaction, heightened repetitive behaviors and spatial memory dysfunction.\\u0026nbsp;\\u003cstrong\\u003eNeurobiological\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003ecorrelates\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eSynaptic ultrastructural abnormalities (presynaptic vesicle depletion, mitochondrial\\u0026nbsp;crista\\u0026nbsp;disorganization) and\\u0026nbsp;\\u003cstrong\\u003edysregulation. Cost efficiency\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eThe protocol\\u0026nbsp;reduces\\u0026nbsp;costs while maintaining phenotypic consistency,\\u0026nbsp;increasing\\u0026nbsp;accessibility for resource-limited laboratories.\\u003c/p\\u003e\\n\\u003cp\\u003eResearch Contributions.\\u0026nbsp;\\u003cstrong\\u003eMethodological\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003einnovation\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eThis study pioneers chronopharmacological ASD modeling, aligning VPA exposure with critical\\u0026nbsp;neurodevelopmental\\u0026nbsp;windows to improve temporal precision.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical and Practical Advancements:\\u0026nbsp;\\u003c/strong\\u003eBy minimizing maternal toxicity and teratogenicity, the protocol adheres to the \\\"3R\\\" principles (reduction, refinement, replacement), setting a new standard for ethical animal research.\\u0026nbsp;\\u003cstrong\\u003eResource Optimization:\\u0026nbsp;\\u003c/strong\\u003eLaboratories in developing regions can leverage cost-saving\\u0026nbsp;designs\\u0026nbsp;to scale ASD research without compromising quality.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eLimitations and Future Directions:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eModel\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003egeneralizability\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eCurrent validation is limited to C57 mice; future studies should test the protocol in rats and\\u0026nbsp;nonhuman\\u0026nbsp;primates to assess cross-species applicability.\\u0026nbsp;\\u003cstrong\\u003eIntegration with\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003emultiomics\\u003c/strong\\u003e\\u003cstrong\\u003e:\\u0026nbsp;\\u003c/strong\\u003eCombining this model with single-cell RNA sequencing or metabolomics could uncover novel biomarkers and mechanistic pathways.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by grants from the Enterprise Joint Fund Project of Hunan Provincial Natural Science Foundation, 2024JJ9097; the Enterprise Fund Project supported by ZYYK; and the GDAS Project of Science and Technology Development (2022GDASZH-2022010110, 2022GDASZH-2022030603-01, 2023GDASZH-2023030602).\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of competing interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors’ contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eZML, CXW and ZYL conceived and designed the study; ZML, CXW, ZXL, LF and HYL performed\\u0026nbsp;the\\u0026nbsp;experiments; ZML and CXW analyzed the data; ZML and CXW visualized the figures;\\u0026nbsp;ZML and CXW wrote the manuscript draft; ZML,\\u0026nbsp;CXW and ZYL revised the manuscript and supervised the study;\\u0026nbsp;all the\\u0026nbsp;authors contributed to the article and approved the submitted version.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll datasets generated and analyzed during this study are presented in this published article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting Interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare\\u0026nbsp;that they have\\u0026nbsp;no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAnimal care and experimental procedures were performed in accordance with the\\u003c/p\\u003e\\n\\u003cp\\u003einstitutional laboratory animal guidelines. The protocols\\u0026nbsp;for the\\u0026nbsp;animal experiments were\\u0026nbsp;as follows:\\u003c/p\\u003e\\n\\u003cp\\u003eapproved by the Institutional Animal Care and Use Committee of Guangzhou\\u003c/p\\u003e\\n\\u003cp\\u003eInstitutes of Biomedicine and Health, CAS (IACUC:2023081).\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eKutluk, G., Kadem, N., Bektas, O. \\u0026amp; Eroglu, H. N. A Rare Cause of Autism Spectrum Disorder: Megaconial Muscular Dystrophy. \\u003cem\\u003eAnn. Indian Acad. Neurol.\\u003c/em\\u003e \\u003cb\\u003e23\\u003c/b\\u003e (5), 694\\u0026ndash;696 (2020).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eGnanavel, S. \\u0026amp; Robert, R. S. 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The critical period of valproate exposure to induce autistic symptoms in Sprague-Dawley rats. \\u003cem\\u003eToxicol. Lett.\\u003c/em\\u003e \\u003cb\\u003e201\\u003c/b\\u003e (2), 137\\u0026ndash;142 (2011).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eWilliams, A. M. et al. Valproic acid improves survival and decreases resuscitation requirements in a swine model of prolonged damage control resuscitation. \\u003cem\\u003eJ. Trauma. Acute Care Surg.\\u003c/em\\u003e \\u003cb\\u003e87\\u003c/b\\u003e (2), 393\\u0026ndash;401 (2019).\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eChoi, C. S. et al. The transgenerational inheritance of autism-like phenotypes in mice exposed to valproic acid during pregnancy. \\u003cem\\u003eSci. Rep.\\u003c/em\\u003e \\u003cb\\u003e6\\u003c/b\\u003e, 36250 (2016).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"ASD model, Valproic acid (VPA), Phase-specific dosing strategy, Maternal toxicity reduction, C57BL/6 mice\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6545657/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6545657/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eValproic acid (VPA)-induced rodent models are widely utilized to study autism spectrum disorder (ASD) but suffer from high maternal mortality (\\u0026gt;\\u0026thinsp;25%) and inconsistent phenotypic outcomes due to imprecise dosing timing and acute embryotoxicity. To address these limitations, we developed a novel triple-phase VPA administration protocol (300\\u0026rarr;400\\u0026rarr;300 mg/kg) in C57 mice, strategically aligned with critical neurodevelopmental windows (E11.5\\u0026ndash;E13.5). This study aimed to optimize ASD modeling by balancing cost-effectiveness with biological fidelity while minimizing maternal toxicity. Behavioral assessments (open field, three-chamber social test, Morris water maze), ultrastructural analysis (TEM), and neuroinflammatory/oxidative stress profiling were conducted to validate model robustness. The results demonstrated that the triple-phase regimen achieved 100% maternal survival (vs. 75% in traditional single-dose protocols, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and significantly reduced abortion rates (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). The offspring exhibited ASD core phenotypes, including social deficits (reduction in stranger interaction time, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.0001), repetitive behaviors (increased marble burying, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), and spatial memory impairments (prolonged escape latency P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). Crucially, synaptic ultrastructural pathologies\\u0026mdash;presynaptic vesicle depletion and mitochondrial crista disorganization\\u0026mdash;were observed alongside dysregulated prefrontal IL-6/TNF-α levels (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and oxidative stress markers (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). The protocol reduced costs through optimized dosing while maintaining phenotypic consistency. This work establishes a standardized, ethically compliant ASD model that reconciles economic efficiency with neurodevelopmental validity, offering a transformative tool for mechanistic studies and therapeutic screening.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Triple-Phase VPA Administration in C57 Mice: A Precision Timing Strategy for Cost-Effective ASD Modeling with Reduced Maternal Toxicity\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-08-14 08:03:14\",\"doi\":\"10.21203/rs.3.rs-6545657/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f8718f17-8bc0-4459-8d19-7ddea074eed0\",\"owner\":[],\"postedDate\":\"August 14th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":52935057,\"name\":\"Biological sciences/Neuroscience\"},{\"id\":52935058,\"name\":\"Biological sciences/Zoology\"},{\"id\":52935059,\"name\":\"Health sciences/Neurology\"}],\"tags\":[],\"updatedAt\":\"2026-04-06T06:41:15+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-08-14 08:03:14\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6545657\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6545657\",\"identity\":\"rs-6545657\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}