We previously found that one mechanism for the avascular retina in a rat model of ROP (Penn 50/10 OIR model) was NAD(P)H oxidase-dependent apoptosis.31
In this study, we found pups exposed to repeated oxygen fluctuations followed by supplemental oxygen (i.e., 50/10 OIR+SO)12
rather than room air (Penn 50/10 OIR) had a further increase in NAD(P)H oxidase activation, determined by increased phosphorylation of subunit p47phox46
(). In addition, the inhibition of NAD(P)H oxidase activity in 50/10 OIR+SO reduced IVNV () but was not associated with reduced VEGF, suggesting there are other mechanisms involving angiogenic signaling through NAD(P)H oxidase activation.
NAD(P)H oxidase is the main source of ROS, particularly superoxide radical, released by neutrophils in response to invading organisms.32
Besides neutrophils, macrophages, bone marrow-derived hematopoietic cells,47
and endothelial cells48
can activate NAD(P)H oxidase to release ROS that trigger disparate signaling events from endothelial cell survival, proliferation, and angiogenesis to endothelial apoptosis.33
It is thought that the concentration and location of ROS released by different degrees of NAD(P)H oxidase activation may trigger different signaling pathways.33,49,50
Our data and previous studies31
provide evidence of this. Here we show that supplemental oxygen (50/10 OIR+SO) further increased NAD(P)H oxidase activation over that in 50/10 OIR and is implicated in angiogenesis (, ), whereas previously we found NAD(P)H oxidase activation in the 50/10 OIR was a cause of peripheral avascular retina through increased apoptosis.31
We also found that the activation of NAD(P)H oxidase and signaling of JNK was triggered in retinal microvascular endothelial cells exposed to hypoxia compared with room air (, ). Previous studies reported that JNK signaling is implicated in H2
and stress-related apoptosis, whereas ERK activation occurred in VEGF-induced endothelial cell survival.51
We also found that the inhibition of NAD(P)H oxidase significantly reduced tissue hypoxia after supplemental oxygen () but was not associated with concomitant reductions in avascular/total retina area (a source of tissue hypoxia6
) or of VEGF (). HP is given systemically 90 minutes before euthanatization and is soluble, quickly diffusing into tissues and cells, forming insoluble conjugates where oxygen tension is lower than 10 mm Hg.52
Binding to oxygen is essential, and the question as to whether redox conditions overwhelm the binding of HP to proteins in low oxygen was disproved in several studies. In the pericentral region of the liver, which has a high concentration of nitroreductase enzymes, binding of HP was assessed during anterograde and retrograde perfusion of the liver. Retrograde perfusion shifted HP binding to the then hypoxic periportal region.52
In addition, high concentrations of NADH or NADPH were shown not to overwhelm the binding of HP to hypoxic tissue.53
HP has also been correlated with other tissue features associated with hypoxia, including spatial relation to perfused vessels and inversely to proliferation54,55
and correlation with oxygen microelectrode measurements when tumor hypoxia was intentionally manipulated.56
All experimental groups in our study were treated in the same manner, and all tissue was processed within the same time frame. Although excess HP is conjugated after death and gives some background signal, the 90-minute exposure time before euthanatization causes the amount of background staining to be less than 1% of the intensity of specific binding on euthanatization. That we did not find differences in avascular/total retina may indicate that measuring the percentage of the retinal avascular area is not as sensitive as measuring conjugated proteins within hypoxic retinal cells. We therefore cannot rule out that hypoxia was related to a reduction in vascular support to the retina.
The 50/10 OIR retinal flatmounts showed hypoxic retina in the avascular zones and in zones throughout the vascular retina, suggesting that all these regions may be vulnerable to the creation of ROS. In contrast, the P4 rat pup retinal flat-mount, which had peripheral avascular retina, did not have HP staining. We suspect this is because at P4, the hyaloidal circulation supplies oxygen to the retina.
Even though the inhibition of NAD(P)H oxidase activation reduced hypoxic tissue in 50/10 OIR+ SO, we were unable to find evidence that VEGF was affected (). We might have missed the time point in our VEGF analysis, though previous studies showed that VEGF was elevated at P12, P14, and P18 in the 50/10 OIR model.57,58
We suspected mechanisms other than VEGF signaling, such as signaling through ROS or inflammatory cytokines,59,60
are involved in the NAD(P)H oxidase-dependent IVNV we found in 50/10 OIR+SO.
We found reduced VEGF in 50/10 OIR+ SO compared with 50/10 OIR but not reduced tissue hypoxia. Again, we took care to treat eyes equally, and our data include minimal background signal of HP after euthanatization. In the kitten OIR model, Ernest6
reported that once avascular retina occurred from hyperoxia induced vaso-obliteration, breathing oxygen did not result in an increase in preretinal oxygen over the avascular retinal areas, though it did initially cause an increase in preretinal oxygen tension over the vascular areas.6
Berkowitz et al.12
found that supplemental oxygen (SO) given over 6 days after 50/10 OIR in the rat, led to a reduced ΔPO2
or a difference in arterial oxygen levels after breathing 95% O2
, 5% CO2
(carbogen), and 21% O2
. (Carbogen breathing can relax the autoregulatory effect of retinal vessels, preventing the constriction seen in hyperoxia or hypertension.) The reduced ΔPO2
in the group rescued in supplemental oxygen suggested a failure in autoregulatory or perfusion reserve, and it can be interpreted as a dysfunction in constriction of retinal vessels in high oxygen or a failure of dilation in low oxygen. Because these pups also had high levels of systemic arterial PO2
, lower ΔPO2
may indicate higher retinal vascular oxygen concentration during supplemental oxygen, which was also what Ernest found initially over the vascular retina in the kitten study.6
Cells within the inner retina (Müller cells and ganglion cells) sense hypoxia and are the major producers of VEGF.61
Increased oxygen to the retina would reduce the stimulus for VEGF expression. Meanwhile, as photoreceptor development occurs and the metabolic demand of the outer retina increases, more oxygen is required. Recent evidence implicates the photoreceptors in creating oxygen demand that results in features of severe retinopathy seen in models of ROP.62,63
Furthermore, the choroid, which is a main supplier of oxygen in the areas of avascular retina, may not be able to meet the oxygen demand because, unlike the choroid in the adult rat, the choroid in the P15 rat was unable to support increased oxygen tension with supplemental oxygen.64
Therefore, given that the metabolic demand of the photoreceptors is inadequately met by the choroidal and retinal vasculatures, it is possible that supplemental oxygen may increase oxygen tension in the retinal vasculature that has a dysfunctional autoregulatory capacity but that still leads to overall retinal hypoxia.
Thus, in the preterm infant, anatomic factors such as avascular retinal area, history of oxygen exposure, current oxygen exposure, and level of maturation of the photoreceptors may play roles in the retinal outcomes of delayed intraretinal vascularization and IVNV. Our data support that signaling events that lead to pathologic features of ROP, namely the avascular retina and IVNV, can depend on the degree of NAD(P)H oxidase activation from repeated oxygen fluctuations followed by room air or supplemental oxygen. Supplemental oxygen and fluctuations in oxygen can cause features that increase the severity of ROP. These studies help explain some of the complexity of oxygen effects in ROP. More study is required because a reduction in oxygen concentration or broad inhibition of ROS may be detrimental to the preterm infant, whose developing central nervous system requires oxygen and whose immune system and abilities to fight infection are limited.65