A novel angiopoietin-derived peptide displays anti-angiogenic activity and inhibits tumour-induced and retinal neovascularization.

BACKGROUND AND PURPOSE: Pathological angiogenesis is associated with various human diseases, such as cancer, autoimmune diseases and retinopathy. The angiopoietin (Ang)-Tie2 system plays critical roles in several steps of angiogenic remodelling. Here, we have investigated the anti-angiogenic effect of a novel angiopoietin-derived peptide. EXPERIMENTAL APPROACH: Using computational methods, we identified peptides from helical segments within angiopoietins, which were predicted to inhibit their activity. These peptides were tested using biochemical methods and models of angiogenesis. The peptide with best efficacy, A11, was selected for further characterization as an anti-angiogenic compound. KEY RESULTS: The potent anti-angiogenic activity of A11 was demonstrated in a multicellular assay of angiogenesis and in the chorioallantoic membrane model. A11 bound to angiopoietins and reduced the binding of Ang-2 to Tie2. A11 was also significantly reduced vascular density in a model of tumour-induced angiogenesis. Its ability to inhibit Ang-2 but not Ang-1-induced endothelial cell migration, and to down-regulate Tie2 levels in tumour microvessels, suggests that A11 targets the Ang-Tie2 pathway. In a rat model of oxygen-induced retinopathy, A11 strongly inhibited retinal angiogenesis. Moreover, combination of A11 with an anti-VEGF antibody showed a trend for further inhibition of angiogenesis, suggesting an additive effect. CONCLUSIONS AND IMPLICATIONS: Our results indicate that A11 is a potent anti-angiogenic compound, through modulation of the Ang-Tie2 system, underlining its potential as a therapeutic agent for the treatment of ocular and tumour neovascularization, as well as other pathological conditions that are dependent on angiogenesis.

Vascular endothelial growth factor in eye disease.

Collectively, angiogenic ocular conditions represent the leading cause of irreversible vision loss in developed countries. In the US, for example, retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration are the principal causes of blindness in the infant, working age and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor (VEGF), a 40kDa dimeric glycoprotein, promotes angiogenesis in each of these conditions, making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can influence cell proliferation, cell migration, proteolysis, cell survival and vessel permeability in a wide variety of biological contexts. This article will describe the roles played by VEGF in the pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the rationales for targeting other VEGF-related modulators of angiogenesis.

The effect of an angiostatic steroid on neovascularization in a rat model of retinopathy of prematurity.

PURPOSE: The inhibition of angiogenesis by angiostatic steroids has been demonstrated in a variety of systems, including rabbit and rat cornea. There is considerable interest in the therapeutic potential of this class of compounds for angiogenic ocular conditions such as diabetic retinopathy, macular degeneration, and retinopathy of prematurity (ROP). This study was designed to test the capacity of an angiostatic steroid, anecortave acetate, to inhibit retinal neovascularization using a rat model of ROP and to investigate the mechanism of the effect. METHODS: At birth, rats were placed in an atmosphere of varying oxygen that produces retinal neovascular changes that approximate human ROP. The rats then received intravitreal injections of either anecortave acetate or vehicle at varying times, and all were subsequently placed in room air. Retinas were assessed for plasminogen activator inhibitor (PAI)-1 mRNA level by RNase protection assay at 1, 2, and 3 days after injection and for normal and abnormal blood vessel growth 3 days later. RESULTS: A significant reduction in the severity of abnormal retinal neovascularization was observed in the steroid-treated eyes compared with vehicle-injected eyes in ROP rats, yet the extent of normal total retinal vascular area was not significantly different. The drug had no effect on either retinal vascular area or neovascularization when tested in room air-raised control rats. Drug-injected eyes demonstrated a six- to ninefold increase in PAI-1 mRNA at 1 to 3 days after injection. CONCLUSIONS: This study represents the first therapeutic effect of an angiostatic steroid in an animal model of neovascular retinopathy. Additionally, the induction of PAI-1 indicates a mechanism of action for this class of compounds, and this is a novel finding in vivo. Because anecortave acetate significantly inhibited pathologic retinal angiogenesis in this model, while not significantly affecting normal intraretinal vessels, it holds therapeutic potential for a number of human ocular conditions in which angiogenesis plays a critical pathologic role.

Ambient hypoxia reverses retinal vascular attenuation in a transgenic mouse model of autosomal dominant retinitis pigmentosa.

PURPOSE: Loss of retinal capillaries is an inherent component of late stage autosomal dominant retinitis pigmentosa (ADRP). This study examined the hypothetical role of tissue hyperoxia in this vascular attenuation process and tested the potential of ambient hypoxia to reverse it. METHODS: Transgenic mice expressing a mutant opsin gene with a 3-bp deletion of isoleucine at codon 255/256 were used. This model is characterized by early onset of a rapidly progressing retinal degeneration that by postnatal day (P)20 results in the loss of all but one row of photoreceptor nuclei. At P20 some mice were placed in 12% oxygen until they were euthanatized at P26. The remainder were maintained in normoxia and killed at the same age. Retinas were dissected, stained for ADPase, and flat-mounted. RESULTS: Deep plexus capillary density was significantly different in normoxic normals versus transgenics at 20 days of age (P:

Inhibition of retinal angiogenesis by peptides derived from thrombospondin-1.

PURPOSE: Thrombospondin (TSP)1 is a tumor suppressor with activity that is associated with its ability to inhibit neovascularization. Previous studies have mapped this antiangiogenic activity to the type 1 repeats and the amino-terminal portion of the molecule within the procollagen-like domain. The present study was performed to investigate the ability of TSP-1 and peptides derived from the type 1 repeats to inhibit retinal angiogenesis. METHODS: TSP-1 and peptides with tryptophan-rich, heparin-binding sequences and transforming growth factor (TGF)-beta1 activation sequences were evaluated in two models of retinal angiogenesis: a retinal explant assay and a rat model of retinopathy of prematurity (ROP). RESULTS: Platelet-derived TSP-1 inhibited angiogenesis in both experimental models. Peptides from the native TSP-1 sequence, which contained both the tryptophan-rich repeat and the TGF-beta1 activation sequence, were the most potent inhibitors of endothelial cell outgrowth in the retinal explant assay. In contrast, a peptide containing only the tryptophan-rich, heparin-binding sequence was most active in inhibiting neovascular disease in the rat ROP model. CONCLUSIONS: These results indicate that the type 1 repeats of TSP-1 contain two subdomains that may independently influence the process of neovascularization, and that peptides derived from these type 1 repeats may be promising pharmacologic agents for treatment of retinal angiogenesis.

Evidence for upregulation and redistribution of vascular endothelial growth factor (VEGF) receptors flt-1 and flk-1 in the oxygen-injured rat retina.

There is considerable evidence that vascular endothelial growth factor (VEGF) is important in the pathogenesis of retinal neovascular diseases. The effects of this endothelial cell-specific mitogen are mediated by specific cell surface receptors. In this study we probed for the two VEGF receptors (VEGFRs) known to have highest affinity in the rat--flt-1 and flk-1. Using a well-characterized rat model of the neovascular disease retinopathy of prematurity (ROP), we performed immunohistochemical assays on methacrylate sections of eyes from normal and oxygen-injured animals at the time neovascularization is first observed (16 days of age) and at its peak (day 20). In day 16 room air retinas there was light, diffuse labeling of the inner nuclear layer and outer plexiform layer. In contrast, in 4 of 5 oxygen-injured eyes on day 16, there was specific labeling of small neovascular growths and normal retinal vessels, and the outermost (sclerad) limit of the label had shifted inward to the vitread border of the inner nuclear layer and the inner plexiform layer. Day 20 room air eyes showed a pattern similar to day 16, although with stronger labeling. However, in oxygen-injured eyes on day 20 the labeling pattern had shifted toward the vitreous, with extremely strong labeling of the preretinal neovascular growths. As on day 16 there was also labeling of the inner plexiform layer and the inner portion of the inner nuclear layer, but not the outer plexiform layer. Comparison of VEGF protein immunolabel with both of the VEGFR immunolabels revealed overlap and strong similarity on day 20 in the oxygen-injured eyes. This is the first report of VEGF receptor protein being concentrated in preretinal neovascular growths in a model of ROP. These results lend themselves to further investigation of the roles of VEGFRs in preretinal neovascularization in ROP and other retinal diseases and suggest avenues of research toward therapies using VEGFR antagonists.

Abnormal panretinal response pattern to carbogen inhalation in experimental retinopathy of prematurity.

PURPOSE: Present technologies are not able to determine which retinas are at risk for the development of neovascularization in retinopathy of prematurity (ROP). In this study, the authors evaluated whether a novel magnetic resonance imaging (MRI) method could be used to identify differences between control retinas and those that will develop neovascularization in the newborn rat model of retinopathy of prematurity (ROP). METHODS: MRI and a 2-minute carbogen (95% O2/5% CO2) inhalation challenge (see ref. 11) were used to measure noninvasively the change in the posterior vitreous oxygen tension in specific locations across the full extent of the retina in day-12 rats raised in either room air (control, n = 7) or variable oxygen conditions (experimental ROP, n = 7). The experimental ROP animals were examined 2 days before the onset of neovascularization. RESULTS: In the ROP group, the response to carbogen was lower (P < 0.05) at every distance from the optic nerve than in the control group. Within the ROP group, the vascular midperipheral retinal reaction to carbogen, 1 to 2 mm from the optic nerve, was as low as that from the avascular periphery, 2 to 3 mm from the optic nerve. Although the vascular central retinal response to carbogen, 0 to 1 mm from the optic nerve, was greater than either the vascular midperipheral retina or the avascular periphery in the ROP group, theoretically this difference could be caused by oxygen diffusing from the hyaloidal circulation. CONCLUSIONS: Carbogen-challenge MRI seems to be a useful tool for assessing the risk of retinal neovascularization in the newborn rat ROP model. This MRI method has potential clinical applicability, for example, because effective laser therapy with retinal sparing may be possible if focal photocoagulation, guided by an MRI map, is performed.

The vitreous protein concentration is increased prior to neovascularization in experimental ROP.

PURPOSE: To test the hypothesis that the vitreous protein content is altered prior to the development of neovascularization in an experimental model of retinopathy of prematurity (ROP). METHODS: Newborn rats underwent either a variable oxygen exposure or room air exposure following birth. On day 13 or 14, two days prior to neovascularization, the steady state vitreous and plasma total protein levels in room-air controls or variable-oxygen-exposed newborn rats were determined; similar measurements were also made for control adult rats. RESULTS: There was a significant difference (P < 0.05, 2-tailed t-test) in the vitreous protein concentration between the age-matched control and experimental newborn rats. The protein level in the ROP rat plasma was not significantly (P > 0.05) different from that in the age-matched control animals. The vitreous-to-plasma-protein ratios of both the control and ROP newborn animals were significantly greater (P < 0.05) than that in the adults. CONCLUSIONS: The results of this study demonstrate, for the first time, increased vitreous protein levels prior to the development of neovascularization in the newborn rat model of ROP. In addition, developmental changes in vitreous protein levels were identified. The role of developmental and pathologic alterations in the blood-ocular barriers in this study is discussed.

Impaired hyaloidal circulation function and uncoordinated ocular growth patterns in experimental retinopathy of prematurity.

PURPOSE: To test the hypotheses that, in the newborn rat model of retinopathy of prematurity (ROP), the hyaloidal circulation is functionally impaired and its development is not well coordinated with that of other ocular structures. METHODS: The functional response of the hyaloidal circulation to a carbogen inhalation challenge was noninvasively evaluated using magnetic resonance imaging (MRI) in day 12 rats raised under either variable oxygen conditions (experimental ROP, n = 8) or room air (control, n = 8). A similar MRI examination was performed in separate experiments using either day 18 newborn control rats (n = 3) or adult rats (n = 9). For each experiment, the hyaloidal circulation perfusion response to carbogen, the functional spatial extent of the hyaloidal circulation in vitreous, and the volumes of vitreous and lens were estimated from MRI enhancement maps. RESULTS: The hyaloidal perfusion response to carbogen breathing in the newborn rats decreased as follows: control day 12 > experimental day 12 > control day 18; no measurable hyaloidal function was found in the adult rat. Regression analysis indicated a relatively poorer superior-inferior correlation in the temporal response to carbogen inhalation for the experimental animals than in the control newborn rats. The vitreous volume decreased in control rats as expected (adult rat > day 18 > day 12). Good agreement was found between the MRI-determined adult rat vitreous volume (56 +/- 2 microliters) and that of previous reports. Functional hyaloidal volumes during carbogen breathing were not significantly different (P > 0.05) between day 18, day 12 control, and experimental newborn rats. The ratio of this functional hyaloidal circulation extent volume to vitreous volume was significantly different (P < 0.05) between these groups. Covariance analysis revealed a relatively less coordinated development between the functional hyaloidal volume and the vitreous volume in experimental animals than in age-matched control animals, whereas there was coordinated evolution of the hyaloidal circulation and the lens in all the animals. CONCLUSIONS: Carbogen-enhanced MRI appears to be a powerful new and noninvasive approach for assessing the functionality of the hyaloidal circulation (that is, its ability to respond to a carbogen challenge) and quantitatively comparing the functional hyaloidal extent to other ocular volumes in the same eye during development and during the disease process. Evidence is presented here for the first time that supports the authors' hypotheses that the function of the hyaloidal circulation in experimental ROP is impaired and that the growth of ocular components are less coordinated.

Therapeutic effect of liposomal superoxide dismutase in an animal model of retinopathy of prematurity.

A newborn rat model of retinopathy of prematurity was used to test the hypothesis that a lack of superoxide dismutase contributes to the retinal vaso-attenuation seen during exposure of the animals to hyperoxic conditions. To determine the endogenous superoxide dismutase activity of the retina under hyperoxic conditions, litters of albino rats were placed in either constant 80% ambient oxygen (constant hyperoxia), or placed in 21% oxygen (room air) immediately after birth. Every other day, for 14 days, several rat pups were sacrificed and their retinas removed for the determination of total superoxide dismutase (SOD) activity and manganese-associated SOD activity. An attempt was made to increase retinal SOD activity by intraperitoneal administration of exogenous SOD encapsulated in polyethylene glycol-modified liposomes. Additional litters were exposed to the same oxygen treatments and supplemented twice daily with either liposome-encapsulated superoxide dismutase in saline or liposomes containing saline without SOD. Animals were sacrificed at various time points for the determination of total superoxide dismutase activity and computer-assisted analysis of vessel density and avascular area. Animals raised in an atmosphere of constant 80% oxygen had significantly reduced levels of retinal superoxide dismutase activity through 6 days of life when compared to their room air-raised littermates. At 6 days of age, daily supplementation with liposome-encapsulated SOD had significantly increased retinal superoxide dismutase activity and reduced oxygen-induced vaso-attenuation as evidenced by increased vessel density and decreased avascular area, when compared to littermates exposed to constant hyperoxia that received control liposomes. Superoxide dismutase had no adverse effects on any of the animals regardless of treatment. Tracing experiments demonstrated that liposomes entered the retina and were found in cells morphologically resembling microglia. Delivery of SOD to the retina via long-circulating liposomes proved beneficial, suggesting that restoration and/or supplementation of endogenous antioxidants in oxygen-damaged retinal tissue is a potentially valuable therapeutic strategy.

Impaired retinal function in young labrador retriever dogs heterozygous for late onset rod-cone degeneration.

Xenon-flash d.c.-electroretinograms were recorded from dark adapted, rod-cone degenerate homozygote affected (n = 6), heterozygote carrier (n = 3) and control retinas (n = 4) at 3 and 4 months of age, starting at 0.6 log units below control PII threshold. One log unit higher stimuli were necessary to evoke PII in heterozygote and affected retinas compared to controls. Unique to the heterozygotes, double peaked PII responses that were evoked by -2 log relative units intensity stimulation were significantly (P = 0.028) lower in amplitude than those of controls. PII amplitudes of homozygotes were significantly (P = 0.005) lower in amplitude than those of controls at both ages examined in response to -2 and 0 log relative intensity stimulation. No differences were found in scotopic threshold response amplitudes or times to peak between the three groups. Homozygote affected PII times to peak were significantly (P = 0.005) shorter in relation to controls at -2 log units. Findings suggest that heterozygotes exhibit an impaired retinal function which can be demonstrated at 3 and 4 months in this mutant.

Detection of vascular endothelial growth factor (VEGF) protein in vascular and non-vascular cells of the normal and oxygen-injured rat retina.

Vascular endothelial growth factor (VEGF) is a potent and specific endothelial cell cytokine that can be up-regulated by hypoxia. There is evidence that VEGF is a significant mediator in retinal neovascular diseases and other disorders in which hypoxia is believed to influence the pathogenesis. Here we demonstrate the spatial relationships among areas of retinal non-perfusion, VEGF protein and vascular endothelial cells throughout the retina, and relate these results to cellular distribution of VEGF in cross section. Newborn albino rats were oxygen-injured by cycles of alternating 50% and 10% oxygen for 14 days and then placed in room air. On days 16, 21 and 26, oxygen-injured and control (raised in room air) rats were sacrificed, enucleated and retinas were dissected and fixed for whole mount immunostaining for VEGF or embedding in glycol methacrylate for VEGF immunohistochemistry. Intact eyes taken on days 16 and 20 were processed similarly. Vascular endothelial cells were demonstrated by staining whole-mounted retinas for adenosine diphosphatase (ADPase) activity. Preretinal neovascular growths (i.e., abnormal vessels extending from the retina into the vitreous) were VEGF-positive. There was also a pan-retinal distribution of non-endothelial cells that were VEGF-positive in both room air and oxygen-injured rats, with stronger immunostaining in day 16 oxygen-injured retinas. In cross-section, VEGF staining was confirmed in preretinal growths, normal retinal vessels, cells in the inner nuclear layer (primarily Müller cells) and ganglion cells. Retinas which had been incubated with nonimmune IgG or absorbed anti-VEGF antibody showed little or no staining. In conclusion, we have identified cells of the inner retina which express VEGF. The production of VEGF by these cells--in particular, Müller cells--may promote preretinal neovascularization in oxygen-injured eyes. We have found, moreover, that the combination of immunohistochemistry and ADPase staining of whole mount preparations is a unique and powerful tool for evaluating relationships between presumed areas of retinal ischemia, VEGF (and other cytokines) and retinal blood vessels, within an entire retina. This approach can be used to study any proliferative retinal disorders in which VEGF is a potential component of the pathogenesis.

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.

Elevation of cGMP with normal expression and activity of rod cGMP-PDE in photoreceptor degenerate labrador retrievers.

Cyclic guanosine 3',5'-monophosphate (cGMP) levels were determined in retinas from a strain of Labrador Retrievers with inherited retinal dystrophy manifesting at early stages of retinal differentiation. The cGMP contents of dystrophic retinas of dogs from 1 to 4 months of age (n = 7) were significantly higher (p = 0.001) than in age-matched controls of the same breed (n = 11). Ultrastructure along the vertical retinal meridian was studied in developing retinas and findings were related to those of age-matched wild-type controls of the same breed. Slow central to peripheral progression of degeneration was observed in affected dogs. No differences were found in total cGMP-phosphodiesterase (PDE) activity, in PDE subunit composition as determined by Western blotting of 2-month-old homozygote affected retinas, or in the amino acid sequence deduced from the nucleotide sequence of the PDE beta-subunit as compared to controls. This model of photoreceptor degeneration thus is the first case of an apparent abnormality of cGMP metabolism that is not associated with a defect in the PDE catalytic subunits, and it is also the first reported model not associated with severe developmental abnormalities and rapid degeneration.

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.

Early morphometry of a retinal dystrophy in Labrador retrievers.

Retinal morphometry was assessed in 7 dogs from a colony of Labrador Retrievers with dystrophic retinas at 1,2,3,4 and 18 months of age. Rod outer segment length and outer nuclear layer width were measured in the central, midperipheral and peripheral retina at six locations along the vertical meridian. Early striking regional differences in onset and rate of progression were characteristic for this inherited retinal degeneration. Notably, some areas of the retina developed fully and normally before degenerating. The central parts of the vertical meridians showed slightly disorganized rod outer segments already at 1 month of age and they were significantly shorter than those of control animals at 3 and 4 months (p < 0.01 and p < 0.001, respectively). The rod outer segments of the midperipheral and peripheral regions were, however, comparable to control animals as late as at 4 months of age. At 18 months the rod outer segments of dystrophic animals were significantly shorter in all retinal regions (p < 0.0005). At the age the outer nuclear layer of the dystrophic animals had become significantly thinner than that of control animals in all retinal regions (p < 0.001), indicating a clear visual cell loss. It is reasonable to characterize this as a retinal degeneration having a relatively slow progression, which enhances its relevance to conditions of clinical significance.