Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by communication and behavioral deficits. Growing evidence points to a significant role of the microbiota-gut-brain axis and immune imbalance in ASD pathogenesis. Gastrointestinal problems and gut microbiota dysfunction are frequently observed in individuals with ASD, suggesting that targeting the gut might alleviate symptoms. This study investigates the potential of prebiotics, non-digestible oligosaccharides that promote beneficial gut bacteria, as a therapeutic approach for ASD. Prebiotics produce short-chain fatty acids (SCFAs), improving intestinal barrier function and promoting immune homeostasis. They also influence the serotonergic system and neuroinflammation, suggesting a mechanism for influencing the gut-brain axis. The valproic acid (VPA) mouse model, which exhibits ASD-like behavioral phenotypes and gut abnormalities, was used to test the effects of a prebiotic diet (GOS/FOS) on ASD-related symptoms. This model is sex-specific, with only male offspring showing the ASD-phenotype; thus, only male mice were used in the study. The selected GOS/FOS prebiotic blend has established immunomodulatory properties, making the immune system a potential mediator of the prebiotic's effect on the gut-brain axis.

Several studies have shown a strong correlation between gastrointestinal issues and the severity of ASD. Alterations in gut microbiota composition have been observed in individuals with ASD, and animal studies have demonstrated that microbiota transplantation from individuals with ASD can induce autistic-like behaviors in rodents. This underscores the critical role of the gut microbiome in ASD pathogenesis. Prebiotics, which selectively stimulate the growth of beneficial gut bacteria, have shown promise in modulating the gut-brain axis, impacting neuroinflammation, and improving behavior. However, research on prebiotics in ASD is still in its early stages.

Eight-week-old BALB/cByJ mice were used. Pregnant dams were injected with either VPA (600 mg/kg, sc.) or PBS on gestational day 11. Male offspring were divided into four groups: PBS control diet, PBS GOS/FOS diet, VPA control diet, and VPA GOS/FOS diet. Dietary intervention started at birth and continued throughout the experiment. Behavioral tests (spatial memory and social interaction) were conducted at P30/31 and P48/49. At P50, mice were sacrificed, and samples (intestine, spleen, blood, brain) were collected. Analyses included Western blotting (TMEM119, CD68), iDISCO and light sheet microscopy (Tph2, Iba1), immunohistochemistry (5-HT, ZO-1), flow cytometry (Th cell subsets), ELISA (haptoglobin), SCFA analysis, and 16S rRNA gene sequencing for microbiome analysis. Statistical analysis involved two-way ANOVA, Tukey's multiple comparisons test, ADONIS test, and Spearman's rank correlation.

The prebiotic diet significantly improved social interaction and spatial memory in VPA-exposed mice at both early and later time points. VPA exposure increased TMEM119 expression (microglial activation) in the cerebellum, and the prebiotic diet significantly reduced CD68 expression (activated phagocytic microglia), suggesting reduced neuroinflammation. VPA exposure led to a reduction in serotonergic neurons in the raphe nuclei, which was mitigated by the prebiotic diet. The prebiotic diet reversed the VPA-induced increase in splenic Th17 cells and decreased the Th1/Th17 to Treg cell ratio, indicating improved immune balance. Serum haptoglobin levels were also restored by the prebiotic diet. In the ileum, the prebiotic diet increased the number of 5-HT+ cells and restored ZO-1 expression, indicating improved intestinal barrier integrity. The prebiotic diet altered the bacterial community diversity and composition in the cecum, restoring key bacterial taxa. Several SCFAs were affected by both VPA exposure and prebiotic intervention, with correlations observed between specific SCFAs and bacterial taxa.

This study demonstrates that a prebiotic diet containing GOS/FOS can effectively ameliorate ASD-like behaviors in a VPA mouse model. The improvements in behavior are associated with normalization of gut microbiota composition, reduced neuroinflammation, restored immune homeostasis, and improved gut barrier integrity. The findings support a strong link between the gut-brain axis and ASD pathogenesis, highlighting the potential of targeted dietary interventions in managing ASD symptoms. The immunomodulatory and neuroprotective effects of the prebiotic diet underscore the importance of the gut microbiome in influencing brain function and behavior. Future studies could investigate the specific mechanisms underlying these effects and explore the efficacy of prebiotic interventions in human populations with ASD.

This study provides compelling evidence for the therapeutic potential of a GOS/FOS prebiotic diet in mitigating ASD-like symptoms in a mouse model. The observed improvements in behavior, immune function, and gut integrity strongly support the gut-brain axis's role in ASD. These results encourage further investigation into dietary interventions as a potential treatment strategy for ASD. Future research should focus on larger-scale studies and human clinical trials to confirm these findings and optimize prebiotic interventions for ASD.

This study used a VPA-induced mouse model, which does not fully replicate the complexity of human ASD. Only male mice were included, limiting the generalizability of the findings to female ASD. Repetitive behaviors, a core symptom of ASD, were not assessed. The long-term effects of the prebiotic intervention were not investigated beyond the timeframe of the study.