Effects of physiologically relevant alcohol concentrations on early retinal development: A microfluidic retinal organoid chip analysis

ABSTRACT: Fetal alcohol spectrum disorder can lead to aberrant retinal development. However, traditional culture systems fail to avoid alcohol volatilization, leaving the pathogenic mechanisms underlying low-concentration alcohol-induced retinal developmental abnormalities at physiological levels still unclear. In this study, we established a long-term low-concentration (0.03% and 0.1% for 3 weeks) and short-term intoxicating-concentration (0.2% for 24 hours) alcohol exposure model based on microfluidic human retinal organoid chip. We found that long-term 0.1% alcohol exposure leads to reduced proliferation, elevated apoptosis, and severe disruption of the structural organization of the neural retina in human retinal organoids. Using single nuclear RNA sequencing (snRNA-seq), we identified a population of abnormal neurons as HuC/D+/Brn-3A- neurons. SnRNA-seq combined with experimental validation revealed a significant increase in this population following alcohol treatment. Furthermore, we identified that impaired energy metabolism in retinal progenitor cells represents the primary mechanism underlying this aberrant differentiation. Alcohol exposure (0.03%) caused a milder phenotype. Additionally, we observed that short-term exposure to intoxication concentrations of alcohol induced developmental delay in human retinal organoids. Notably, Microelectrode Array recordings further demonstrated that alcohol treatment significantly impaired the functional maturation of human retinal organoids. This study identifies impaired energy metabolism as a key driver of alcohol-induced aberrant neurogenesis in human retinal development, providing a mechanistic basis for exploring therapeutic interventions for fetal alcohol spectrum disorder-associated retinal lesions. It also confirms that the microfluidic retinal organoid chip serves as a reliable platform for the toxicological investigation of volatile substances represented by alcohol.