Promising Repetitive unidirectional Spinal Tactile Stimulation Promotes Microglial Synaptic Modulation in mPFC of ASD Mouse through Bmal1

Background Synaptic abnormalities are hallmark pathological features of autism spectrum disorders (ASD), contributing to the behavioral impairments frequently observed in these neurodevelopmental conditions. Microglia, as the brain’s primary immune cells, are essential for synaptic refinement during adolescent development. Disrupted microglial-mediated synaptic pruning has been implicated in pathophysiology of ASDs, however, the underlying mechanisms remain incompletely elucidated. In this context, repetitive unidirectional spinal tactile stimulation (RSTS) has emerged as a promising non-invasive therapeutic strategy. By delivering gentle, unidirectional tactile stimulation to the skin surface over spinal region, RSTS has been shown to modulate microglial function and effectively restore synaptic balance. Objective This study aims to explore how RSTS enhances microglial synaptic pruning in the medial prefrontal cortex (mPFC) during adolescent development in ASD mice, with a specific focus on the role of Brain and Muscle ARNT-Like 1 ( Arntl1 ), a core circadian protein crucial for regulating this process. Methods ASD mice underwent RSTS treatment during adolescent brain development for 21 days, administered twice daily for 10 minutes per session. Behavioral changes were evaluated using the three-chamber social interaction and open field tests. Synapse number and morphology were assessed through Golgi staining. To determine the therapeutic effects of RSTS during adolescent brain development, microglial synaptic pruning and synaptic protein expression were analyzed using immunofluorescence staining and Western blot. Furthermore, the molecular mechanism underlying the synapse pruning implications of RSTS in ASD during adolescent were comprehensively investigated using single-nucleus RNA sequencing (snRNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). Finally, the role of Bmal1 was validated in Bmal1 knockout mice, confirming its involvement in the enhancement of microglial synaptic pruning by RSTS during adolescent brain development in ASD. Results RSTS was found to alleviate autistic-like behaviors in adolescent ASD mice during brain development. Results from snRNA-seq and ChIP-seq analyses indicated that the therapeutic effects of RSTS may be mediated through microglial Bmal1 and its role in the transcriptional regulation of microglial synaptic pruning. Furthermore, in vivo experiments confirmed that RSTS enhances microglial synaptic pruning in mPFC of adolescent ASD mice via Bmal1. These findings suggested that Bmal1 serves as a critical target of RSTS in facilitating microglial pruning during the adolescent brain developmental period in ASD mice. Conclusion This study represents the first comprehensive investigation into the underlying mechanisms of RSTS in treating ASD, utilizing single-cell sequencing, gene-knockout mice, and complementary molecular analyses. Our findings suggest that the therapeutic effects of RSTS are potentially mediated through the modulation of Bmal1 -dependent microglial synaptic pruning and the regulation of key synaptic proteins and the complement system. These results provide novel empirical evidence for the role of RSTS in restoring synaptic balance and offer valuable insights into its potential as an intervention for ASD, further elucidating the regulatory pathways through which Bmal1 contributes to neurodevelopmental disorders.