This is the final paper in a series reporting our studies on the effect of hyperoxia on the retinal vessels of the rabbit. It deals with the in vitro behaviour of the adult andimmature retinal vascular complex and of growing endothelial cells in tissue culture when exposed to anoxic, hypoxic and hyperoxic atmospheres. Comparison is made with growth in air, described in the previous paper, and the influence of added carbon dioxide and of changes in temperature and pH is reported. An account is given of the materials and methods employed, including time-lapse cinephotomicrography, phase-contrast light microscopy, conventional light microscopy and electron microscopy.
Immature retinal capillaries exposed to pure oxygen showed, after 4–6 hr, shrinkage of the whole complex and individual capillary retraction. Movement of macrophages became sluggish and after 8–12 hr exposure the endothelial cells became swollen and broke down into granular debris. Within 24–30 hr the complex had entirely disintegrated: no growth occurred. Comparable experiments with 10% oxygen showed less breakdown than in air and outgrowth was profuse. Mature retinal capillaries exposed to pure oxygen showed much greater resistance to hyperoxic injury.
Growing cells from the capillaries exposed to pure oxygen showed arrest of growth and contraction after 4–5 hr exposure; disintegration of cells began to appear after 18–22 hr and by 48 hr cellular death was complete (that is, some hours later than the immature capillaries). Macrophages in the culture were somewhat more resistant. This effect was obtained in all concentrations of oxygen above 30% at which level injury was minimal. Cells grew well in 10% oxygen and for short periods in pure nitrogen, but with cellular abnormalities and, in the case of the latter gas, with variable degrees of degeneration after 72-hr exposure.
The ultrastructural evolution of the oxygen injury is described. Granular degeneration is largely due to the formation of autophagic vacuoles and it is thought that lysosomal injury is an important early change.
These findings are discussed and related to cognate work in the literature. It is concluded that the reactivity of retinal vascular endothelium to oxygen in vitro is closely comparable to that of retinal vessels in vivo, both in animals and man, showing a uniform mode of cellular breakdown and parallel relationships between the degree of cellular maturity, the period of exposure and the concentration of oxygen. It follows that injury to the retinal endothelium in all these circumstances is most probably due to the direct action of oxygen.