Effect of a water-soluble vitamin E analog, trolox C, on retinal vascular development in an animal model of retinopathy of prematurity.

The debate over the efficacy of vitamin E as a therapy for retinopathy of prematurity (ROP) continues 45 years after it was first proposed. The discrepancies between one clinical study and another may be due to the difficulty of delivering a lipid-soluble molecule like vitamin E to the immature retina. Trolox C is a water-soluble analog of vitamin E with potent antioxidant activity. We have studied the effectiveness of intraperitoneal injection of Trolox C in an animal model of ROP. Albino rats were placed in 80% oxygen at birth where they remained for 14 d before sacrifice and assessment of retinal vasculature. Rats were administered 625 microg/kg Trolox C, or vehicle, by intraperitoneal injection on alternate days for the duration of the exposure. Other rats were simultaneously raised in room air, injected, and assessed as controls. Percent avascular retinal area, vascular leakage, and retinal capillary density were measured by computer-assisted image analysis. Trolox C-injected rats had significantly smaller avascular areas (14.6 +/- 4.8% vs. 25.4 +/- 6.3%), less leak area (0.04 +/- 0.07 mm2 vs. 0.16 +/- 0.14 mm2), and greater capillary density (24.3 +/- 2.6 pixel % vs. 18.9 +/- 3.1 pixel %) than vehicle-injected counterparts. These findings indicate that Trolox C facilitated the process of retinal vasculogenesis under hyperoxemic conditions. They also suggest that oxygen free radical-mediated damage plays a role in the pathologic effect of high oxygen rearing of newborn rats. Additional studies are warranted to determine precise site(s) and mechanism(s) of Trolox C activity in this and similar disease models in which peroxidation is believed to play a causal role.

Long-term retinal vascular abnormalities in an animal model of retinopathy of prematurity.

PURPOSE: A conventional criticism of animal models of retinopathy of prematurity (ROP) concerns the common occurrence of rapid spontaneous resolution of retinal vascular sequelae. The purpose of this study was to determine whether animals subjected to a novel variable oxygen exposure protocol would undergo the rapid spontaneous resolution of retinal vascular pathology that is typical of past models. METHODS: Newborn rats were exposed to an oxygen environment that alternated between 50% and 10% every 24 h for 14 days and then removed to room air, or were raised from birth in room air as controls. To determine early retinal vascular growth rate, both exposed and non-exposed rats were sacrificed between 3 and 28 days of age, after which eyes were enucleated and retinas dissected and stained for adenosine diphosphatase (ADPase) activity to demonstrate the vasculature. Rats were maintained in room air for 2 to 18 weeks after the variable oxygen exposure period for assessment of long-term retinal vascular abnormalities by ADPase histochemistry. RESULTS: The retinal vasculature of oxygen-exposed rats was significantly different from that of room air-raised rats with respect to capillary density, branching frequency, and bifurcation angle. These differences were restricted to the area that was vascularized after removal to room air (the peripheral-most 25% of the retinal area), and they persisted for the duration of the study. CONCLUSIONS: We have developed a rat model of ROP using an exposure protocol designed to create systemic oxygen levels that approximate those of premature infants. This model does not demonstrate the complete resolution of vessel abnormalities that historically has limited animal models of ROP.

The range of PaO2 variation determines the severity of oxygen-induced retinopathy in newborn rats.

PURPOSE: This study was conducted to determine the potential influence of PaO2 fluctuation on the retinal neovascular response known to occur in newborn rats exposed to hyperoxic conditions. As an inherent corollary, the authors also defined the relationship between the fraction of inspired oxygen (FiO2) and the arterial blood oxygen tension (PaO2) in newborn rats. METHODS: Experiment 1 was composed of several oxygen-exposure protocols in which atmospheres of 10% oxygen concentration were alternated with different higher levels of ambient oxygen (50%, 40%, 30%, and room air). In experiment 2, two alternating oxygen concentrations were made to converge toward room air (20.9% oxygen) with each successive group of four treatment groups. These included another group exposed to alternating 50% and 10% oxygen, a group exposed to alternating 45% and 12.5% oxygen concentrations, one exposed to alternating concentrations of 40% and 15% oxygen, and a final group exposed to 35% and room air oxygen concentrations. In each case, oxygen was alternated between the two exposure concentrations every 24 hours. The term delta FiO2 is used to designate the difference in the two oxygen concentrations to which a treatment group was subjected, applying the units of fraction of inspired oxygen (i.e., delta FiO2 = 0.4 for the exposure to alternating 50% and 10% oxygen). At birth, litters of albino rats were placed in each of these environments for 13 or 14 days, after which PaO2 and retinal vascular development were assessed in some rats. The remainder were removed to room air for 4 days before the incidence and severity of abnormal neovascularization were measured. RESULTS: PaO2 and FiO2 were directly and linearly correlated (r2 = 0.998). In experiment 1, the extent of retinal vascular development on removal from oxygen was a linear function of delta FiO2. Retinal neovascularization subsequently occurred in all rats exposed to alternating 50% and 10% or 40% and 10% oxygen concentrations, but only a third of the 30% and 10% exposure group, indicating a minimum threshold for proliferative disease at delta FiO2 = 0.2. In experiment 2, retinal avascularity also increased linearly with increasing delta FiO2. There was a threshold for neovascularization between the exposure to alternating 45% and 12.5% oxygen and the 40% and 15% oxygen exposure (100% versus 4.8% incidence of neovascularization), indicating a requirement of < or = 12.5% oxygen episodes to stimulate a consistent proliferative response. CONCLUSIONS: These results suggest that PaO2 fluctuation and degree of hypoxia may have more influence on proliferative retinal disease in newborn rats than the extended hyperoxia that has historically received greater attention. Experimental designs that address the inherent differences in pulmonary function between intrinsically healthy animals and compromised premature infants are of substantial value to our understanding of the pathogenesis of retinopathy of prematurity.

Exposure to alternating hypoxia and hyperoxia causes severe proliferative retinopathy in the newborn rat.

Exposure to variable hyperoxia has recently been shown to be much more effective at producing proliferative retinopathy in the newborn rat than exposure to constant hyperoxia. To incorporate a more clinically relevant oxygen-exposure paradigm in our studies, we have now used a cycle between 50 and 10% oxygen and have compared its effects with those found using new exposures to the previously used 80/40% cycle. Starting at birth and continuing for 14 d, rats were exposed to environments that cycled between 50 and 10% oxygen or 80 and 40% oxygen every 24 h. After exposure, some rats were killed for assessment of retinal vascular development. Others were removed to room air for 4 d before killing and evaluation for the presence of abnormal neovascularization--a clinical consequence believed to be promoted by termination of oxygen therapy. The 50/10% cycle resulted in greater retardation of retinal blood vessel development during oxygen than that found in the 80/40% exposure group. After 4 d postexposure in room air, the incidence of preretinal neovascularization was 97% in the 50/10% rats and 72% in the 80/40% group. Clearly, the overall amount of oxygen the subject receives is less critical than other parameters of its administration in producing proliferative retinopathy. Also, the range of variation (40% in both cases) is not the controlling characteristic. Our results suggest that consistency of oxygen level and avoidance of hypoxic levels should be important concerns in neonatal oxygen therapy.

Oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization.

PURPOSE: To confirm a relationship between oxygen-induced retinal vasoattenuation and subsequent abnormal neovascularization in the newborn rat. METHODS: Beginning at birth, some litters of Sprague-Dawley rats were exposed to 80% constant oxygen while others received oxygen varying between 40% and 80% in a cyclic fashion. The frequency of the change in inspired oxygen (FiO2) was either 6, 12, 24, or 48 hours. The exposures periods lasted for 14 days, at which time some rats from each exposure group were sacrificed and assessed for retinal vasoattenuation with injection of fluorescein-labeled dextran. The remaining rats from each group were transferred at day 14 from the hyperoxic atmosphere to room air for an additional 4 days. These animals were then killed and assessed for retinal neovascularization by staining for vascular ADPase activity. RESULTS: Of all rats raised in variable oxygen, 62% exhibited abnormal retinal neovascularization after 4 days in room air. Only 18% of the rats exposed to constant oxygen responded with abnormal neovascularization. Among the four groups of variable oxygen-exposed rats, there was a direct correlation (R2 = 0.96) between degree of retinal avascularity upon removal from oxygen and the propensity for subsequent abnormal neovascularization. Constant oxygen-exposed rats did not exhibit this relationship. This exposure produced the greatest retinal avascularity upon removal from oxygen but the lowest incidence of abnormal neovascularization after 4 days in room air. CONCLUSIONS: Retinal avascularity may not be the single overriding stimulus for neovascularization in oxygen-induced retinopathy. Other hypotheses bear consideration, including the possibility that variable oxygen leads directly to vascular endothelial cell mitosis, a common retinal manifestation of ischemia-reperfusion.

Variable oxygen exposure causes preretinal neovascularization in the newborn rat.

PURPOSE: To test the hypothesis that variable hyperoxia potentiates preretinal neovascularization in newborn rats, and to establish a more reliable animal model of ROP in which therapies designed to inhibit abnormal angiogenesis can be tested. METHODS: Immediately after birth, litters of Sprague Dawley albino rats and mothers were placed in an incubator containing 40% oxygen. After 12 hours, the oxygen was increased to 80% with a transition time of less than 1 min. For the ensuing 7, 10, or 14 days, the oxygen was altered between 40% and 80% every 12 hr in a stepwise fashion. Other litters were kept in constant 80% oxygen or in room air for the same three time periods. After exposure, rats were either killed or placed in room air for an additional 2, 4, or 7 days before being killed. RESULTS: When rats were killed immediately after oxygen exposure, the resulting vessel loss in rats exposed to 40%/80% oxygen was identical to that of animals exposed to 80% (vessels constituted 12.2 +/- 2.2% of total retinal area in cyclic oxygen vs 12.0 +/- 1.2% in constant oxygen). However, preretinal neovascularization subsequently occurred in 66% (63/96) of all rats exposed to cyclic oxygen followed by a room air period but in no rats (0/50) exposed to constant oxygen followed by room air. Preretinal vascular proliferation consisted of glomerular tufts of endothelial cells, or mature, lumenized vessels containing red blood cells. CONCLUSIONS: Consistency of oxygen therapy is more important than overall oxygen level in inducing retinopathy. Consideration should be given to tighter control of intended oxygen therapy in premature infants, regardless of the target saturation level.

Oxygen-induced retinopathy in the rat: hemorrhages and dysplasias may lead to retinal detachment.

Rearing neonatal rats in hyperoxia induces the development of retinal hemorrhages and retinal dysplasia. Albino rats were placed in 80% oxygen immediately after birth and were exposed for either 5, 10, or 14 days, followed by sacrifice or exposure to normoxia for an additional 2, 4, 5, 7, 8, 10, 38, 45 or 56 days. Control rats were simultaneously raised in room air and sacrificed at the same times. All animals were enucleated and their eyes processed for light and electron microscopy. Eyecups were trimmed to facilitate cross-sectioning of the retina in the vertical meridian. No control rats showed signs of retinal hemorrhages or of dysplastic folds or rosettes. Nor did the retinas of rats killed immediately after oxygen exposure contain hemorrhages, but the incidence of retinal folds or rosettes in this group was 54%. For rats exposed to combinations of hyperoxia and brief normoxia (10 days or less), 40% suffered hemorrhages and 50% developed retinal folds or rosettes. Although hemorrhages were more prominent in rats subjected to longer periods of oxygen (73% of all rats exposed for 14 days followed by brief normoxia vs. 6% of those exposed for 5 days followed by brief normoxia), the incidence decreased with time post-exposure in room air. Hemorrhages occurred in 100% of the rats raised in oxygen for 14 days followed by 2 days in room air, and decreased to 50% by 7 days in room air and to 0% by 38 days, indicating a spontaneous resolution with time. In each case, the blood appeared to leak from the newly-forming vessels of the deep capillary net, with most of the red blood cells migrating to the subretinal space. Retinal fold or rosette formation, indicative of developmental dysplasia, occurred in a fraction of virtually all groups of exposed rats, and persisted at the longest post-exposure periods. These two manifestations of oxygen-induced retinopathy are emphasized because they lead to an abnormal separation of the retina from the epithelial layer, which may increase the likelihood of the most serious consequence of ROP--retinal detachment. In fact, all rats that endured post-exposure periods of 38 days or longer before sacrifice exhibited retinal detachment.

Effect of light history on the rat retina: timecourse of morphological adaptation and readaptation.

Sprague Dawley rats were born and raised under either 5 or 800 lux cyclic light (12L:12D) and were sacrificed at 1, 2, 3, 6, 12, 16, and 28 weeks of age. At each time point outer nuclear layer (ONL) area and rod outer segment (ROS) length were measured. The former is an estimation of photoreceptor number, and the latter is an estimation of the photon-catching integrity of the retina, both of which are known to be dependent on the light environment. Regression analysis revealed an ONL area reduction with time of 0.003 mm2/wk for 5-lux-reared rats and 0.009 mm2/wk for 800-lux-reared rats. ROS length was relatively constant in the dim light group, but showed a decline in 800 lux rats of 0.5 microns/wk. Rats moved from 800 to 5 lux at 9 and 21 wks of age showed no significant change in ONL area after 3 wks. ROS length in these rats increased at a prodigious rate, and in the 12-wk-olds (9 wks at 800 lux, followed by 3 wks at 5 lux), ROS length exceeded that of age-matched rats raised in 5 lux for life.

Predisposing factors to light-induced photoreceptor cell damage: retinal changes in maturing rats.

Retinal changes occurring during the period of growth and maturation of Long Evans pigmented rats were examined to obtain a better understanding of the basis for the age-dependency of light-induced photoreceptor cell damage. Susceptibility to light damage increased markedly between 30 and 60 days of age and to a lesser extent between 60 and 90 days. Although the retinal antioxidant vitamins E and C, and taurine showed a significant increase during the age-period studied, retinal lipid phosphorus and total protein increased by similar amounts indicating that the concentration of these nutrients was not changing. In contrast, rhodopsin content of the retina increased progressively by 44% between 30 and 90 days of age. While ROS length showed no appreciable change with age, rhodopsin per ROS length increased by 31% between 30 and 60 days of age and by 48% between 30 and 90 days. Determinations of ROS phospholipid to rhodopsin ratio and disks per ROS length indicated that rhodopsin did not become more concentrated in photoreceptor cells between 30 and 90 days. However, the 12% increase in ROS diameter between 30 and 90 days of age may partially account for the rhodopsin difference. These findings demonstrate an age-dependent association between greater rhodopsin per ROS length and increased susceptibility to retinal light damage. An increased metabolic demand on photoreceptor cells with greater rhodopsin may be an important factor influencing their destruction by light.

Separate mechanisms for retinal damage by ultraviolet-A and mid-visible light.

Retinal damage by light has two distinct action spectra, one peaking in the ultraviolet-A (UVA) and the other in the mid-visible wavelengths (green light). Here we show in a single animal species, the Long Evans rat, that UVA and green light can produce histologically dissimilar types of damage. UVA light in particular produces severe retinal damage at low irradiation levels. Furthermore, the mechanism of damage by UVA light is different from that of green light as determined by their relative rhodopsin bleaching efficacies. These results provide convincing evidence that different chromophores mediate damage by UVA and green light. By producing both classes of damage in a single species, a sound model is provided for further investigation into the different forms of photic retinopathy.