in contrast, shh signaling, which is involved in cell differentiation and tissue patterning, may be more sensitive to metabolic perturbations with a more dynamic regulation, which can lead to its downregulation under glucose fluctuations treatments. however, despite the observed upregulation of shh and sox2 in ng mio - m1 cells, the expression of rhodopsin, a marker of mature neuronal cells, was not detected. this indicates that, although the cells possess the potential to differentiate into neuronal cells, the conditions employed in our protocol were not sufficient to fully induce this differentiation. the lack of mature neuronal marker expression is likely due to the timing and duration of the experimental conditions. it may therefore be worthwhile to optimize the protocol in order to more ac curate ly reflect the diabetic conditions in humans, with a view to promoting full neuronal differentiation. given the role of shh in muller cells reprogramming and the observed upregulation under sustained high - glucose and gf treatments, future experiments could investigate the effect of exogenous shh treatment on human muller cells. this approach would provide insights into the therapeutic potential of modulating shh signaling to mitigate the adverse effects of reactive gliosis and enhance regenerative processes in the diabetic retina. although the current study provides important insights into the gliotic and reprogramming potential of muller cells under different glucose treatments, some limitations should be considered. the study employed the mio - m1 cell line, which, although well established in human retinal research, may not fully capture the physiological complexity of muller cells found in human retina. future research could benefit from using primary muller cells to better reflect the in vivo context of human diabetic retinopathy. in addition, the effects of other stressors, such as oxidative stress or hypoxia, which also play a role in diabetic retinal damage, were not included in our model. inclusion of these factors could provide a more comprehensive understanding of the cellular mechanisms involved. despite these limitations, the current study contributes important insights into the gliotic and reprogramming potential of muller cells under varying glucose conditions. statistical analysis all the results are expressed as the mean ± sem ( standard error of the mean ) of at least three independent experiments. statistically significant differences were assessed using prism 6. 05 ( graphpad prism software, inc., la jolla, ca, usa ) with student ’ s t - test for statistical comparison between groups. differences between means were considered statistically significant when p - values were at least * * * 5. conclusions * * * the results showed that mio - m1 muller cells exhibit distinc t responses to different glucose treatments, which are strongly dependent on their metabolic environment. this differential response could have implications for the onset and progression of diabetic retinopathy. the increased levels of activation and dedifferentiation markers observed in ng mio - m1 cells in response to different glucose treatments suggest a protective response that may occur in early or well - controlled diabetes. in contrast, the lack of response observed in hg mio - m1 cells exposed to different glucose treatments may reflect an exhausted gliotic and reprogramming capacity, which could contribute to the development and progression of diabetic complications such as retinal neurodegeneration and diabetic retinopathy observed in uncontrolled diabetic patients exposed to prolonged metabolic glucose stress. although our results provide valuable insights, further in - depth studies are needed to elucidate the mechanisms involved and to explore the potential therapeutic implications for mitigating retinal neurodegeneration associated with diabetes. extending these studies to primary muller cells in future research would provide a more physiologically relevant system, helping to validate and refine our findings. this approach would provide a deeper understanding of the cellular response to sustained high - glucose and glucose fluctuations and could improve the development of targeted therapeutic strategies for retinal pathologies in human patients.