Activation and reprogramming of human Mu?ller cell line MIO-M1 exposed to high glucose and glucose variability: an in vitro study

Background and aim. In diabetic subjects, retinal neurodegeneration is associated with reactive gliosis which involve the activation of Mu?ller cells (MCs), mainly characterized by increased expression of glial fibrillary acidic protein (GFAP). Recently, in lower vertebrates, it has been reported that, following reactive gliosis, MCs acquire characteristics of retinal stem cells and that Sonic Hedgehog (SHH) is a crucial regulator of this de-differentiation process. Although diabetic condition causes reactive gliosis in the retina, the effect of glycemic fluctuations on activation and reprogramming of human MCs is yet unknown. The aim of the study is to assess the activation and reprogramming of the human Mu?ller cell line MIO-M1 exposed to high glucose (HG) and glucose variability (GV). Materials and Methods. Human Muller cell line MIO-M1 was cultured in a medium containing either 5 mM (N cells) or 25 mM of glucose (H cells) and then incubated for 96 h in a medium with: basal glucose (5 or 25 mM), high glucose (25 or 45 mM), basal glucose and high glucose alternated every 24 hours (5/25 or 25/45 mM), basal glucose and single exposure to high glucose in the last 24h (5/s25 or 25/s45 mM), low glucose and high glucose alternated every 24 hours (3/25 or 5/45 mM). MCs activation and reprogramming was studied through the expression of GFAP and SHH respectively, evaluated by western blot analysis. In addition, a morphological analysis of MCs was performed by using GFAP and SHH immunolabelling on micrographs obtained with a fluorescence microscope. Results. In N cells cultured with normal glucose (5mM), an upregulation of GFAP expression was observed in response to HG and GV conditions. Moreover, this increase was associated with morphological changes and a higher number of hypertrophic cells. In H cells cultured with high glucose (25mM), there was no modulation of GFAP expression in response to either treatments. In N cells, an upregulation of SHH was also observed in response to HG and GV treatments, with a peculiar localization of the protein in multiple spots inside the cytoplasm. On the contrary, H cells showed a reduced expression of SHH in response to the treatments without morphological changes of intracellular SHH localization. Conclusion. Our results highlight activation and reprogramming features of human Muller cell line MIO-M1 cultured in normal glucose and exposed to HG and GV conditions.