Abstract
Purpose::
In vertebrate eyes, the choriocapillaris is thought to provide oxygen and nutrients to the outer retinal layers. In mammals including humans, additional blood vessels enter the retina from the vitreal side and ramify in the plexiform layers. However, in other vertebrates including chicks, such vessels are absent. Earlier studies indicated that as the retina matures, it loses mitochondria from the inner portion, and converts to anaerobic glycolysis as a source of energy. We have attempted to determine the oxidative state of different portions of the developing retina using an immunohistochemical approach.
Methods::
EF5, a pentafluorinated derivative of etanidazole, can react with reduced proteins to form conjugates. Exposure of tissues to EF5, followed by immunolocalization with ELK3-51, a highly specific antibody, can indicate levels of hypoxia within the tissues. Regions of high EF5 reaction in chick retina of different developmental ages were visualized using Cy3 conjugated to ELK3-51. Avidin-Alexa 488, which can specifically bind to biotin containing carboxylases in the mitochondria, was used to localize these structures.
Results::
As development of the chick retina proceeds, different oxidation states in different layers were found. For E(mbryonic day) 5 chick retina, the staining was almost evenly distributed throughout the tissues. For E7 chick retina, the hypoxic stain was concentrated in a subset of ganglion cells. For E11 chick retina, the hypoxia is observed in the inner portion of the inner nuclear layer and ganglion cell layer. For E14 chick retina, hypoxia was observed at the ganglion cell layer, and additional reaction was seen in the inner portion of the inner nuclear layer and the outer plexiform layer. There was also some reaction in the region of budding photoreceptors. For E19 chick retina, the Ganglion cell layer, and the inner nuclear layer were more hypoxic. The Pecten oculi is a corrugated flap of pigmented, vascularized tissue that extends along the back of the eye and is embedded in the vitreous body of the avian eye. The oxidative state of the pecten oculi was also examined with EF5/Cy3. The pecten oculi was found much less hypoxic than adjacent retina tissue.
Conclusions::
EF5 staining can be an effective approach to analyze the oxidative state of the developing retina. These studies indicate that there is a complex interaction between potential sources (Choriocapillaris, Pecten Oculi), and cellular consumers of dissolved oxygen.
Keywords: retinal development • microscopy: light/fluorescence/immunohistochemistry • immunohistochemistry