when applicable, definition of asterisks and descriptive measures were indicated in the figure legends or results. drawing of the chemical structure of apg - 1387 shown in figure 1 was done using the chemdraw software. discussion muller glial cells play fundamental roles in retinal tissue functions. therefore, studies into their biology and functions may contribute to understanding the causes of retinal pathologies and to develop strategies to alleviate their outcomes. the aim of this study was to investigate the impact of high glucose and glucose fluctuations on critical cellular processes, such as gliosis and reprogramming in muller cells. these processes are of pivotal importance in the onset and progression of retinal neurodegeneration and diabetic retinopathy. in the present study, we observed a significant increase in gfap expression in ng mio - m1 cells exposed to sustained high - glucose and gf treatments, indicating a glucose stress - induced gliotic response. this response was associated with other markers of reactive gliosis, such as the morphological changes associated with the transition from a bipolar to a radial morphology and the overexpression / reorganization of vimentin intermediate filament. these findings indicate that ng mio - m1 cells are sensitive to glucose changes in their surrounding environment, potentially functioning as a protective or reactive mechanism in response to metabolic stress. the remarkable sensitivity to glucose stress and the prompt reactive gliosis observed in mio - m1 cells were found to be similar to those observed in muller cells of diabetic subjects [ 8 ]. this finding confirms the suitability of mio - m1 cells as an in vitro model to study muller cell response. in contrast, hg mio - m1 cells maintain a basal high level of both gfap and vimentin together to less organized intermediate filaments across the different glucose treatments, suggesting a limited and static response to sustained high - glucose and gf treatments. these findings highlight that mio - m1 cells in chronic hyperglycem ic conditions may adapt or develop enhanced tolerance over time due to constant and prolonged exposure to high glucose levels. the adaptation or desensitization of hg mio - m1 cells to sustained high - glucose and gfs may indicate a saturation of the gliotic response, which is characteristic of prolonged hyperglycem ic states, as observed in uncontrolled diabetes [ 34 ]. this adaptation or desensitization may result in a reduction in the cellular capacity to respond to additional metabolic challenges or stress. to understand the clinical consequences of muller cell gliosis it is essential to also consider the reprogramming process, which may include dedifferentiation of muller cells and regeneration of various retinal neurons, contributing to retinal tissues repair under certain conditions. in species such as birds [ 35 ], zebrafish [ 36, 37 ], and rodents [ 38 ], muller glia contribute as the primary source of retinal regeneration. when the retina is damaged, muller glia can dedifferentiate into muller glia - derived progenitor cells ( mgpcs ), acquiring a progenitor - like phenotype and starting to proliferate, thus contributing to retinal repair [ 39, 40 ]. notably, muller glia in lower vertebrates exhibit a remarkable ability to regenerate retinal neurons, contrasting with the limited regenerative capacity seen in mammals, including humans [ 40 ], where muller cells typically respond to injury with reactive gliosis, leading to scarring rather than regeneration [ 11, 20 ]. the shh signaling pathway is essential for the proper development of all vertebrate retinas [ 41, 42, 43, 44, 45, 46, 47 ], and several studies have demonstrated the involvement of shh signaling in the proliferation and differentiation of mgpcs, contributing to retinal regeneration in lower vertebrate [ 18, 19, 31, 32, 34 ]. however, in mammals, the shh pathway alone may not be sufficient to overcome the intrinsic limitations of muller cells to regenerate neuronal cells. in our study, we observed an increase in shh expression in ng mio - m1cells exposed to sustained high - glucose and gf treatments. this suggests that these cells are engaging in pathways associated with cellular dedifferentiation and potential reprogramming towards a progenitor state. the increased shh protein expression in ng cells was accompanied by significant changes in the intracellular localization of shh, exhibiting a more spotted / punctate pattern in the cytoplasm and in close proximity to the plasma membrane. these findings suggest an enhanced state of cellular activity with increased synthesis and potential accumulation of shh, aligning with the findings in lower vertebrates and rodents, where increased shh signaling plays a role in muller cell reprogramming and retinal regeneration [ 18, 48, 49 ]. furthermore, significantly higher levels of shh were observed in hg mio - m1 cells compared to ng mio - m1 cells. conversely, hg mio - m1 cells exposed to gf treatments showed a decrease in shh protein expression levels. a trend toward a decrease was also observed in response to sustained high - glucose. this finding is consistent with a previous study in which the authors observed a decrease in shh signaling in reactive astrocytes of the cerebral cortex after acute, focal injury, particularly in cells proximal to the lesion site [ 50 ]. this highlights that the negative regulation of shh activity in astrocytes is context - dependent and varies with the degree of cellular damage. in our study, the decreased shh levels observed in hg mio - m1 cells exposed to gfs indicate that the severity of metabolic stress may influence shh signaling pathways through a similar mechanism. these observations underscore the notion that glial cells, including muller cells and astrocytes, exhibit differential shh responses depending on the extent of glucose stress, highlighting the dynamic regulation of shh signaling in glial cells. although the shh level has decreased, the cells that express it maintain a dot - like distribution of shh inside the cells. our findings suggest that, under ng condition, exposure to sustained high - glucose and gfs stimulates shh expression, potentially promoting the dedifferentiation and reprogramming of muller cells. however, in hg - adapted cells, additional metabolic stress from gf treatments leads to decreased shh expression, possibly impairing regenerative capacity and enhancing gliotic responses. these observations indicate that the severity and fluctuation of glucose stress influence shh signaling pathways, affecting the balance between neuroprotection and gliosis in the retina. a previous study demonstrated that high - glucose conditions have been associated with increased sox2 levels in human muller glial cells, which may support cell survival and regeneration under stress conditions [ 51 ]. in our experiments, sox2 was significantly upregulated in hg mio - m1 cells, compared to ng mio - m1 cells. furthermore, ng mio - m1 cells have the capacity to modulate this gene when exposed to sustained high - glucose and gf treatments, exhibiting a consistent upregulation of sox2. these findings are consistent with the observed upregulation of shh in ng mio - m1 cells exposed to different glucose treatments. unlike ng mio - m1cells, hg mio - m1 cells exposed to the same glucose treatments do not show variations in sox2 expression. this different behavior of hg mio - m1 cells, with regards to sox2 and shh expression, may be due to the different roles and regulatory mechanisms in the glial cells of these two genes. sox2 is a transcription factor crucial for maintaining stemness and promoting cell progenitor proliferation, and its upregulation could be a response to cellular stress in order to maintain or enhance regenerative capacity.