It is important to acknowledge certain limitations in our study. Notably, our experimental approach relied on a two-dimensional cell culture, which, while valuable for investigating microglial responses and gene expression patterns, does not recapitulate the complex three-dimensional interactions that occur in the brain and in vivo. The specific mechanisms by which microglia detect and respond to ethanol are still not fully understood. Moreover, it should be recognized that cultured cells incubated with ethanol do not fully replicate microglia in humans with chronic alcohol consumption. Further investigations using brain organoid models (32) or in vivo (30) studies will help gain a more comprehensive understanding of the dynamics and functional consequences of microglia-neuron interactions in AUD and the role of genetic factors in these interactions. Our current study did not specifically investigate the mechanisms of interaction between genetic risk factors and environmental influences, which are crucial in determining an individual’s susceptibility to AUD. Polygenic and environmental risks often reinforce each other (82). Factors such as family dynamics, peer interactions, adverse life events, and broader societal factors such as religion and education interact
