Oxidative damage is the result of oxidative insults due to the production of reactive oxygen species and reactive nitrogen species generated by local ischemia. Accordingly, such increases in oxidative protein damage can affect cellular integrity . In the present study, we evaluated whether the expression of oxyproteins was increased in the optic nerve head after ET-1-induced optic nerve ischemia. The number of cells in the retinal ganglion cell layer and inner and outer nuclear layers, as well as the thicknesses of the cell layers, decreased over time. The thickness of the inner and outer plexiform layer was also decreased. These results suggest that ischemia-induced oxidative stress can damage both the retina and optic nerve.
The term ‘oxidative stress’ is used to indicate excessive increases in the levels of reactive oxygen and nitrogenous compounds compared with normal physiological intracellular levels . The eye in particular is exposed to light, radiation, oxygen, and chemicals. In addition, noxious reactive oxygen/oxygen-derived free radicals are produced normally during cellular metabolism of oxygen through processes such as electron hopping and oxidation-reduction [14, 15, 31]. As such, various antioxidative defense mechanisms have evolved to protect cells from oxidative damage [31, 32]. The oxides produced via physiological metabolism are eliminated by antioxidants, and damaged DNA and proteins are repaired. However, some structural abnormalities resulting from excessive oxidative stress may be beyond the capacity of physiologic recovery mechanisms .
Excessive free radicals in a cell can disrupt intracellular structures, including nucleic acids, lipids, and proteins [34, 35]. Furthermore, proteins are the primary target of oxygen-derived free radicals [36–39]. An excess in reactive oxygen species induced by oxidative stress directly triggers insults in the retina, including mitochondrial dysfunction,  induction of apoptosis, increased neurotoxin production, weakening of the neuroprotective functions of glial cells, and activation of immune-mediated neuronal injuries. Indeed, oxidative stress in retinal ganglion cells and retinal proteins may be the cause of optic disc deformities [10, 11].
Quantification of intracellular oxyproteins is necessary for a comprehensive understanding of oxidative stress. Measurement of DNPH protein bound carbonyl derivatives is one of the most commonly used strategies to quantify oxyproteins, and DNPH is quite sensitive with respect to immunodetection using specific antibodies . In our study, following two weeks of treatment with ET-1, the expression of oxyproteins was significantly increased compared to the control group. However, at four and eight weeks, the level of oxyprotein expression was equal to or slightly decreased compared to the control group. When a protein undergoes irreversible oxidative modifications and cannot be repaired, it is eliminated through degradation. The mechanisms of protein elimination are through either proteome-mediated proteolysis  or chaperone-mediated autophagy . In either case, vulnerable cells damaged by ischemia and oxidative stress may have their damaged proteins eliminated by these mechanisms, thereby allowing resistant cells to persist. Thus, equivalent levels of oxyprotein expression at four and eight weeks suggests the presence of a relative increase in stressful environments as well as expression of oxidative proteins in the ET-1- administered group compared to the control group.
The results of the present study also demonstrate the presence of oxidative stress in the control group. Specifically, apoptosis was more apparent in the control group than in the ET-1-treated group, which may be a reflection of the effects of natural stress such as light, chemicals, or radiation. Furthermore, the basal production of reactive oxygen species, which are regulated by antioxidants, is part of normal cellular redox homeostasis. Thus, the balance between native and oxidatively-damaged proteins depends on the rates of protein biosynthesis, oxidative modification, and oxidized protein elimination .
In our results, there was no expression of oxyproteins in the retina by oxyblot, yet cell loss was observed in retinal layers. In this study, we analyzed separation of the optic nerve head from the retinal layer. For this analysis, we collected optic nerve head tissue, which protrudes into the globe. The optic nerve head consists mainly of the surface nerve fiber layer and the prelaminar layer. Those layers include mainly axons and some deep retinal layers. The structure of the retina containing whole layer is different from that of the surface retinal layers. Therefore, if oxyprotein is expressed more in the axons, the oxyblot result will be different from that of the thin axons of the existing retinal layers. Since oxyblot is a quantitative method, a certain amount of oxyprotein is needed for detection. Therefore, the absence of detection can be attributed to a low amount of oxyprotein in the tissue. To date, there have been no studies regarding whether oxyprotein can migrate along the axon. In a previous animal model study, the injection of ET-1 into the posterior chamber caused a significant constriction of retinal vessels and impaired retrograde axonal transport by decreasing the number of retinal ganglion cells . The number of retinal ganglion cells decreased by 44.2% four weeks after the injection of ET-1 into the posterior chamber. For this reason, apoptosis in retinal ganglion cells should be rarely observed in the ET-1-treated group.