Neurodevelopmental disorders (NDDs) encompass a wide range of conditions, including intellectual disability, ADHD, and ASD, significantly impacting social, cognitive, and behavioral functioning. The prevalence of ASD and ADHD is rising, underscoring the need to understand their etiology. While genetic factors play a role, environmental factors also contribute significantly, with evidence suggesting that over 50% of new ASD cases are attributed to non-genetic factors. Traffic-related air pollution (TRAP) has emerged as a potential environmental risk factor, with epidemiological studies linking proximity to heavily trafficked roads during gestation and early life to increased NDD risk. However, these studies often suffer from confounding variables and lack precise exposure quantification. Animal models offer a valuable tool to establish causality and investigate the effects of TRAP on NDD-relevant behaviors. Previous preclinical studies have limitations in their translational relevance due to factors like repeated anesthesia and simplified exposure methods. This study utilized a novel real-time rodent exposure facility to address these limitations, exposing developing rats to real-world TRAP conditions from a traffic tunnel, while controlling for noise and vibration. Rats were chosen due to their complex social behaviors. The study examined the impact of this exposure on physical growth, neonatal reflexes, communication, social interaction, and learning/memory.

A substantial body of epidemiological research has linked exposure to traffic-related air pollution (TRAP) and proximity to busy roadways with an increased risk of neurodevelopmental disorders (NDDs). Studies have utilized various methods and populations, reporting associations between TRAP exposure, particularly during critical developmental windows (late gestation and early childhood), and diagnoses such as autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD). However, these epidemiological studies often struggle to isolate the effects of TRAP from other confounding factors, including socioeconomic status, parental smoking habits, and nutritional differences. The difficulty lies in accurately quantifying individual exposure to complex environmental mixtures. The Childhood Autism Risks from Genetics and Environment (CHARGE) study, for instance, highlighted a significant correlation between proximity to freeways (within 309 meters) and autism risk, particularly during the third trimester of pregnancy. This existing epidemiological evidence emphasizes the need for preclinical studies to establish a direct causal link and explore the underlying mechanisms connecting TRAP exposure to NDD development. While some preclinical research exists, many studies employ simplified exposure methods that lack the translational relevance of real-world scenarios.

Pregnant Sprague-Dawley rat dams were divided into three groups: (1) a laboratory control group housed at a standard vivarium; (2) a roadside exposure group transported to a facility adjacent to a freeway tunnel, with dams randomly assigned to either TRAP (traffic-related air pollution) or FA (filtered air) exposure chambers; (3) a transport control group, where dams were transported halfway to the roadside facility and back to control for transport stress. Offspring (two males and two females per litter) underwent behavioral testing from postnatal day (PND) 4 to 50. The behavioral assays included: assessment of developmental milestones (body weight, length, reflexes); isolation-induced pup ultrasonic vocalizations (USVs); juvenile reciprocal social interaction; open-field exploration; novel object recognition; and contextual and cued fear conditioning. The roadside exposure facility simulated near-roadway conditions, drawing air directly from the tunnel face for the TRAP group and using a multi-stage filtration system for the FA group. Noise and vibration were minimized through building insulation and vibration isolators. Data were analyzed using appropriate statistical methods, including ANOVA, t-tests, and the Log-Rank test. Control groups were used for all behavioral tests to account for potential confounds. Litter effects were accounted for in the experimental design and analysis.

Near-roadway exposure, regardless of whether the offspring were exposed to TRAP or filtered air (FA) at the roadside facility, resulted in significant developmental delays. Offspring in both roadside groups showed delayed development of psychomotor reflexes compared to the laboratory control group. Open field locomotion revealed abnormal activity patterns in both roadside groups. The TRAP group exhibited the lowest number of isolation-induced 40-kHz pup ultrasonic vocalizations, indicating impaired communication. TRAP exposure also affected some aspects of social communication, reflected in reduced neonatal pup ultrasonic calling and altered juvenile reciprocal social interactions. The transport control group did not show significant behavioral differences from the laboratory control group, suggesting that transport stress was not a primary factor influencing behavioral outcomes. Importantly, both roadside groups (TRAP and FA), with exposure to the same noise and vibration stress, showed consistent behavioral effects, indicating that the air pollution component of near-roadway exposure plays a major role in the observed neurodevelopmental alterations.

This study provides compelling evidence for a causal link between near-roadway exposure and neurodevelopmental alterations in juvenile rats. The findings demonstrate that both TRAP exposure and the non-air pollution components of near-roadway environments (noise and vibration) contribute to behavioral deficits. The consistent impact on several key aspects of development, including psychomotor reflexes, vocalizations, and social interaction, underscores the far-reaching effects of early life exposure. The observed delays in developmental milestones and alterations in social communication are consistent with the behavioral phenotypes observed in neurodevelopmental disorders, supporting the epidemiological findings that link proximity to traffic with increased NDD risk. The fact that the filtered-air group at the roadside facility also exhibited behavioral changes highlights the need to consider all aspects of near-roadway environments, not just air pollution, when assessing potential health impacts. Further research is needed to elucidate the mechanisms underlying these effects and to determine the specific contributions of individual pollutants in the TRAP mixture.

This study demonstrates a strong association between developmental exposure to near-roadway conditions, including traffic-related air pollution and related noise and vibration stressors, and the manifestation of neurodevelopmental deficits in juvenile rats. These findings strongly support the growing epidemiological evidence linking near-roadway residence to increased neurodevelopmental disorder risk. Future research should focus on identifying the specific mechanisms through which these environmental stressors affect neurodevelopment, including investigating the roles of individual pollutants and characterizing the long-term consequences of early life exposure. Furthermore, studies are needed to translate these preclinical findings to humans and identify effective interventions to mitigate these risks.

While this study utilized a novel and translationally relevant exposure model, some limitations exist. The study focused on specific behavioral domains, and the effects of near-roadway exposure on other aspects of neurodevelopment should be further explored. The study used Sprague-Dawley rats, a specific strain; the generalizability of the findings to other rat strains and species warrants investigation. Although the facility controlled for noise and vibration, it may not perfectly replicate the complexity of human near-roadway environments. Furthermore, the specific chemical composition of the TRAP exposure may vary over time.