Development Of Oxygen-induced Retinopathy Research Articles

Melatonin and its bioisosteres as potential therapeutic agents for the treatment of retinopathy of prematurity.

We conducted a study on the impact of intraperitoneal injections of melatonin and its three bioisosteres (compounds 1-3) on the development of oxygen-induced retinopathy in newborn rats during a 21-day experiment. It was demonstrated that melatonin and its analogues 1-3 effectively reduce the total protein concentration in the vitreous body of rat pups, decrease concentration of VEGF-A, and lower the level of oxidative stress (as indicated by normalization of antioxidant activity in the vitreous body). Melatonin and its analogues 1-3 equally normalize the level of VEGF-A. Analogues 1 and 2 even exceed melatonin in their ability to reduce protein influx into the vitreous body. However, analogue 2 had no effect on antioxidant activity, while analogues 1 and 3 caused a significant increase in this parameter, with analogue 3 even slightly exceeding melatonin. Thus, it can be concluded that analogues 1-3 are comparable to melatonin and can be utilized as potential therapeutic agents for the treatment of retinopathy of prematurity.

The effect of conditional knocking out vascular endothelial growth factor gene on the mouse model of oxygen induced retinopathy

Objective To observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR). Methods The conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Muller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF- 1α) was detected by immunofluorescence analysis. Results During the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59), the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer. Conclusions The local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability. Key words: Microglia; Mice, knockout; Vascular endothelial growth factors; Disease models animal; Hypoxia-inducible factor 1, alpha subunit

Hypoxia-hyperoxia paradigms in the development of oxygen-induced retinopathy in a rat pup model.

Retinopathy of prematurity [ROP] continues to be a significant clinical problem in preterm infants. There is a need for animal models to better understand the roles of hypoxia/hyperoxia in the pathogenesis and management of ROP. To test the hypothesis that multiple daily cycles of intermittent hypoxia, followed by brief hyperoxia, would provide a clinically relevant protocol for generation of ROP in a rat pup. Rat pups were exposed for the first 14 days to one of three protocols: room air [RA], sustained cycles of hyperoxia/hypoxia [SHH] as previously employed to produce ROP in rat pups, and intermittent hypoxia/hyperoxia [IHH] in order to more closely simulate clinical conditions in preterm infants. Retinae were obtained at 18 days and imaged for both avascularization and neovascularization. As expected, the SHH group demonstrated significantly increased avascularity [40.9 ± 7.9% of retina] which was minimal in both RA and IHH groups. All SHH exposed pups exhibited neovascularization which occurred in 5/7 IHH exposed retinae versus 0 in the RA group [p = 0.02]. However, mean number of clock hours of neovascularization after IHH was 1.9 ± 2.1 which did not differ from the RA group, and was less than in the SHH group [8.3 ± 1.9, p < 0.001]. A more clinically relevant intermittent hypoxia/hyperoxia [IHH] protocol does not produce the same degree of ROP as the traditional sustained hypoxia/hyperoxia [SHH] paradigm. Nonetheless, further refinement of our model may provide a suitable model for understanding the lesser degrees of ROP which predominate in preterm infants.

HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

BackgroundHypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.Methodology/Principal FindingsWe investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Conclusions/SignificanceBoth HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Exposure to cyclic oxygen sufficient for development of oxygen-induced retinopathy does not induce bronchopulmonary dysplasia in rats

ABSTRACTBronchopulmonary dysplasia and retinopathy of prematurity affect premature infants exposed to supplemental oxygen. Susceptibility to oxygen-induced retinopathy in the rat is heritable, with inbred Dark Agouti (DA) rats being more susceptible than Fischer 344 (F344) rats. To establish if hyperoxic exposure sufficient to induce florid retinopathy would induce strain-specific lung changes, newborn DA and F344 rats were exposed to cyclic hyperoxia or room air for up to 18 days. Lung function was assessed at 18 days, and standardized morphometry and immunohistochemistry were performed at intervals. No differences in arterial blood gases or protein concentration of bronchoalveolar lavage fluid were observed amongst groups at 18 days, although lung wet-to-dry weights were significantly lower for F344 than for DA rats. Pulmonary vascularity increased in all oxygen-exposed animals compared with room air-exposed controls, but there was no significant difference between strains. The lung surface area of oxygen-exposed F344 rats was significantly increased at day 10 compared with F344 controls and oxygen-treated DA rats, but at 14 and 17 days the oxygen-exposed DA rats showed increased lung surface area compared with oxygen-exposed F344 rats. The minor morphological differences found in the lung did not affect pulmonary function, suggesting that mechanisms inducing oxygen-induced retinopathy and bronchopulmonary dysplasia are fundamentally different, and that susceptibility to bronchopulmonary dysplasia is not heritable in the rat.

Interleukin-18 regulates pathological intraocular neovascularization

Recently, the proinflammatory cytokine IL-18 has been shown to have a role in angiogenesis. This study aimed to elucidate its role in abnormal neovascularization (NV) in an oxygen-induced retinopathy (OIR) mouse model of the retinopathy seen in human premature newborns. IL-18 was constitutively expressed in the retina in C57BL/6 mice, but expression transiently dropped on Day 17 after birth in mice exposed to 75% oxygen for 5 days between Days 7 and 12. Coincident with the IL-18 reduction in oxygen-treated mice, vascular endothelial growth factor was expressed in the retina, and OIR developed. By Day 24, NV in the retina had regressed to normal levels. By contrast, IL-18 knockout mice, exposed to elevated oxygen concentrations, developed more severe OIR on Day 17, and it is important that this persisted until Day 24. This suggested that IL-18 negatively regulated retinal NV. To investigate this further, we administrated recombinant IL-18 to C57BL/6 mice during the development of OIR but found no significant inhibition of retinopathy. However, when IL-18-binding protein was administered during the OIR recovery phase to neutralize endogenous IL-18, OIR was still apparent on Day 24. We therefore concluded that IL-18 regulates pathogenic retinal NV by promoting its regression rather than inhibiting its development. This suggests some useful, new approaches to treating retinopathy in humans.

Dexamethasone Alters TNF-α Expression in Retinopathy

TNF-α has been found in the retina. Hyperoxia and hypoxia regulate TNF-α expression. TNF-α is an important factor in inflammation and angiogenesis. Dexamethasone inhibits TNF-α production. Changes in TNF-α expression in the retina may play an important role in the development of oxygen-induced retinopathy. Oxygen-induced retinopathy was produced in C57BL6 mice by exposure to 75% oxygen at Postnatal Day 7 (P7) for 5 days and the mice recovered in room air until Day 17 (P17). Dexamethasone was administered at 0.5 mg/kg/day once daily subcutaneously during the 5 days of oxygen exposure. TNF-α expression was evaluated at Day 7 prior to oxygen exposure, at Day 12 (P12) immediately upon removal from oxygen, and at Day 17, the time of maximal vasoproliferation by RT-PCR. TNF-α is developmentally regulated in the retinae of C57BL6 mice. From P7 to P12, there is a 3-fold increase in TNF-α expression and from P7 to P17 there is a 2.7-fold increase. There was 2.7-fold suppression in expression immediately following oxygen exposure at P12. The expression was dramatically increased at P17, the time of maximal vasoproliferation. Dexamethasone inhibited the expression of TNF-α at P17 by 6.4-fold. At this dose, it also suppressed the baseline TNF-α expression in the mouse model. In summary, TNF-α is altered in the development of oxygen-induced retinopathy in the mouse. It increased markedly during the vasoproliferative phase and was suppressed by dexamethasone. Modulation of TNF-α expression may provide a potential site of action for future therapeutic targets.