Background: The ex vivo model represented by mouse retinal explants in culture is a useful experimental model to investigate the molecular mechanism involved in neurovascular diseases such as diabetic retinopathy (DR). It ensures an experimental overview with more complete respect to isolate cells and reduce problems in terms of accessibility and management with respect to in vivo model. In particular, it allows the evaluation of the relationship between retinal cells in response to the typical stressors involved in DR pathogenesis.

Methods: Ex vivo retinal fragments derived from 3- to 5-week-old C57BL/6J mice. In particular, after dissection, the retina is cut into 4 separate fragments and transferred onto inserts placed with ganglion cells up. Once in culture, the explants could be treated in stress conditions typical of DR. In particular, this study protocol describes the procedure for the preparation and the culture of retinal explants with specific metabolic stressors such as high glucose (HG), advanced glycation end product (AGE), and oxidative stress (OS). In the end, this paper provides the protocols to perform molecular analyses in order to evaluate the response of retinal explants to stress and/or neuroprotective treatments.

Discussion: The cultured retinal explants represent an ex vivo experimental model to investigate the molecular mechanisms involved in neurovascular diseases such as DR. Moreover, they could be useful to test the effect of neuroprotective compounds in response to metabolic stressors in a fewer time respect to an in vivo model. In conclusion, retinal explants in culture represent a valuable experimental model to conduct further studies to better understand the pathophysiology of DR.