Abstract
ABSTRACTHypoxia plays an important role in several retinal diseases, especially in central retinal artery occlusion (CRAO). Although CRAO has been known for over a hundred years, no cure or sufficient treatment is available. Potential therapies are being evaluated in several in vivo models or primary cultures. However, in vivo models or primary cultures are very time-consuming, expensive, and furthermore several therapies or agents cannot be tested. Therefore, we aimed to develop a standardized organotypic ex vivo retinal hypoxia model. A chamber was developed in which rat retinal explants were incubated for different hypoxia durations. Afterwards, the retinas were adjusted to normal air and incubated for 24, 48 or 72 h under standard conditions. To analyze the retinal explants, and in particular the retinal ganglion cells (RGC) immunohistology, western blot and optical coherence tomography (OCT) measurements were performed. To compare our model to a standardized degeneration model, additional retinal explants were treated with 0.5 and 1 mM glutamate. Depending on hypoxia duration and incubation time, the amount of RGCs decreased and accordingly, the amount of TUNEL-positive RGCs increased. Furthermore, β-III-tubulin expression and retinal thickness significantly decreased with longer-lasting hypoxia. The reduction of RGCs induced by 75 min of hypoxia was comparable to the one of 1 mM glutamate treatment after 24 h (20.27% versus 19.69%) and 48 h (13.41% versus 14.41%) of incubation. We successfully established a cheap, standardized, easy-to-use organotypic culture model for retinal hypoxia. We selected 75 min of hypoxia for further studies, as approximately 50% of the RGC died compared to the control group after 48 h.
Highlights
Hypoxia plays an important role in several ophthalmologic diseases such as central retinal artery occlusion (CRAO) (Hayreh et al, 2004), anterior ischemic optic neuropathy (AION) (Hayreh, 2009a,b), glaucoma (Schmidl et al, 2011; Cherecheanu et al, 2013), retinal vein occlusion (Terui et al, 2011; Hayreh, 2005), and diabetic retinopathy (Wessel et al, 2012)
Chamber-characteristics at 37°C The here presented autoclavable full-metal chamber was the third generation of chambers we designed for this hypoxia model
The partial oxygen pressure inside the full-metal chamber was reduced to 0.1% by N2influx (Fig. 2B). 5 min after sealing the chamber the partial oxygen pressure rose to 0.2%
Summary
Hypoxia plays an important role in several ophthalmologic diseases such as central retinal artery occlusion (CRAO) (Hayreh et al, 2004), anterior ischemic optic neuropathy (AION) (Hayreh, 2009a,b), glaucoma (Schmidl et al, 2011; Cherecheanu et al, 2013), retinal vein occlusion (Terui et al, 2011; Hayreh, 2005), and diabetic retinopathy (Wessel et al, 2012). In the mentioned diseases retinal hypoxia/ischemia results in irreversible visual impairment and blindness (Osborne et al, 2004). Since the RGC-5 cell line dedifferentiated (Van Bergen et al, 2009; Schnichels et al, 2012b; Schultheiss et al, 2012; Wood et al, 2010; Krishnamoorthy et al, 2013) only the use of primary RGCs remains for cell culture experiments. The preparation of primary RGCs is both very time- and animal-consuming, the complexity of a whole organ system is lacking and the RGCs are extracted out of their natural tissue network. The cells in a RGC primary cell culture are hardly comparable with the cells in a living tissue
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