Oxidative stress‐induced retinal damage is prevented by mild hypothermia in an ex vivo model of cultivated porcine retinas

Abstract

Background Hydrogen peroxide (H2O2) can be used in vitro to simulate oxidative stress. In retinal organ cultures, H2O2 induces strong neurodegeneration of the retina. It is known that oxidative stress plays a role in the development of several retinal diseases including glaucoma and ischemia. Thus, we investigated whether processes underlying oxidative stress can be prevented by hypothermia using an ex vivo organ culture model of porcine retinas. Methods Porcine retinal explants were cultivated for 5 and 8 days. Oxidative stress was induced via 300 μM H2O2 on day 1 for 3 hours. Hypothermia treatment at 30°C was applied simultaneously with H2O2, for 3 hours. Retinal ganglion cells (RGCs), apoptosis, bipolar and cholinergic amacrine cells, microglia and macroglia were evaluated immunohistologically. Apoptosis rate was additionally analysed via western blot. Results Reduced apoptosis rates through hypothermia led to a preservation of RGCs (P < .001). Amacrine cells were rescued after hypothermia treatment (P = .17), whereas bipolar cells were only protected partly. Additionally, at 8 days, microglial response due to oxidative stress was completely counteracted via hypothermia (P < .001). Conclusions H2O2 induced strong degenerative processes in porcine retinas. The role of oxidative stress in the progression of retinal diseases makes this ex vivo organ culture model suitable to investigate new therapeutic approaches. In the present study, the damaging effect of H2O2 to several retinal cell types was counteracted or strongly alleviated through hypothermia treatment. Especially RGCs, which are affected in glaucoma disease, were protected due to a reduced apoptosis rate through hypothermia.

Reduced Retinal Degeneration in an Oxidative Stress Organ Culture Model through an iNOS-Inhibitor

“…Interestingly, in our study, 1400 W prevented the H 2 O 2 -mediated increase of Bax + cells. In a previous study, oxidative stress led to an increased amount of Bax [ 6 ]. The application of H 2 O 2 leads to oxidative stress, which results in mitochondrial damage leading to the activation of the intrinsic apoptotic pathway.…”

“…This is in contrast with our previous study, where we detected a loss of amacrine cells after five days. However, in this previous study, the number of cholinergic amacrine cells in H 2 O 2 -stressed retinas was analyzed, whereas in the present one, all amacrine cells were investigated [ 6 ].…”

“…Porcine eyes were obtained from the local abattoir. Preparation of retinal explants was carried out as described previously [ 5 , 6 , 7 , 8 ]. Retinal explants were cultured on inserts (Merck Millipore; Burlington, MA, USA) in a 6-well plate with 1 mL Neurobasal-A-medium (Life Technologies, Carlsbad, CA, USA) supplemented with 0.8 mM·L-glutamine (Life Technologies), 2% B27 (Life Technologies), 1% N2 (Life Technologies), and 2% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA).…”

“…In order to investigate the time course of the cell death mechanisms after iNOS-inhibition, an earlier point in time (four days) was also analyzed, since in previous studies, cell death could already be detected at an earlier point in time in the H 2 O 2 degeneration model [ 6 ] and in a hypoxia degeneration model, protection through the iNOS-inhibitor could already be identified after four days [ 5 ].…”

“…Additionally, research of retinal degeneration occurring in glaucoma, retinal ischemia, or age-related macular degeneration (AMD) is increasingly being carried out in ex vivo organ cultures [ 3 , 4 ]. In recent years, our research groups have adapted retinal organ culture models to investigate the pathomechanisms of retinal diseases and to test new therapeutic options [ 5 , 6 , 7 , 8 ]. These models are based on porcine retinas, which can be obtained as a waste product from the food industry.…”