gms | German Medical Science

Introduction: Recently, hydrogen sulfide (H₂S) has been recognized as a third-gas signaling molecule of great importance to the central nervous system. H₂S shows a highly protective effect on neurons by modulating oxidative stress and inflammatory responses. Retinal ganglion cells (RGCs), an ocular neuronal cell, are highly sensitive to intraocular pressure (IOP). Elevated IOP seems to lead to loss of RGCs by causing an imbalance in iron homeostasis in retina. The aim of this study is to investigate whether elevated IOP causes changes in iron homeostasis leading to RGCs loss in retina and whether H₂S plays a protective role in RGCs damage, resulting from an imbalance in iron homeostasis.

Methods: Equal-aged male C57BL/6 mice (n=24) were executed in a CO₂ atmosphere and retinal explants were harvested. Retinal explants were cultured for 24 hours under fluctuating pressure for the first 6 hours (pressure was increased to 60 mmHg every 15 minutes) with or without the addition of 100nM H₂S donor (GYY4137), or with 500 nM Hemin for the first hour. RGCs were quantified by immunohistochemical staining against Brn3a in flat-mounts. Retinal cryo-sections (12um) were immunohistochemically stained against Transferrin receptor (TfR), H-Ferritin, L-Ferritin and hepcidin and photographed under confocal microscope (LSM 980 Airyscan2, Zeiss), fluorescence intensity was analyzed by Image J. Statistical analysis and presentation was performed by GraphPad Prism. Statistical significance was determined by t-test and one-way ANOVA with Tukey-Kramer's post hoc tests for comparison between groups. Differences were considered statistically significant at p<0.05.

Results: Compared to the control group, iron overload and elevated pressure led to significant loss of RGCs. The number of RGCs was reduced by approximately 20% (p<0.05) and 35% (p<0.05). H₂S protected the RGCs from loss due to elevated pressure. TfR, H-ferritin, L-Ferritin and Hepcidin were expressed in RGCs. Both iron overload and elevated pressure increased the expression of TfR in the RGC layer (p<0.05). In addition, the expression of Hepciden was significantly increased in the retina under elevated hydrostatic pressure (p<0.05). However,when H₂S was added to the culture medium, the expression of TfR and Hepciden were significantly decreased (p<0.05).

Conclusions: RGCs are sensitive to iron toxicity and elevated hydrostatic pressure. H₂S protects the RGCs against elevated pressure. At the same time elevated hydrostatic pressure disrupts iron homeostasis in retina by increasing iron import, reducing iron export, and decreasing the storage of stabilized iron. H₂S precursor protects RGCs lose by promoting iron homeostasis.