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.

Intracellular topography of rhodopsin regeneration in vertebrate rods.

The vertebrate visual pigment of rods, rhodopsin, bleaches in light and regenerates in darkness. When the bleaching and regeneration are carried out in vivo, it is found that the regeneration takes place at nonuniform rates along the rod outer segment (ROS): toads and frogs regenerate rhodopsin faster in the proximal ends of the ROS than in the distal ends. Rats do the reverse. These patterns of regeneration persist whether the bleaching is done with flashes or with steady light. They are also independent of the extent to which the retinal pigment epithelium contains melanin. Furthermore, the dichotomy of patterns (proximal faster vs. distal faster) does not seem to depend upon the presence of an excess of stored retinoid in the eye. Instead, it is suggested that the villous processes of the epithelial cells may play an important role in the regeneration patterns. These processes in amphibia extend nearly to the rod inner segment but in the rat they surround only the apical end of the outer segment. If they "funnel" the retinoids back to the ROS, their location and morphology could explain the two different kinds of patterns seen.

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.

Effects of continuous light on the retina of a fish, Notemigonus crysoleucas.

Notemigonus crysoleucas organisms were exposed to light at 1,250 and 850 lux in regimes of 12L:12D and 24L:OD. Each layer of cells in the retina was evaluated employing light microscopy and measurements for quantitative analysis were recorded from the retinas of over 150 fish. These values were compared for dorsal and ventral portions of the retina, for the two intensities, and for the two regimes. Fish maintained under the 24L:OD light regime exhibited significant light damage to the retina, characterized by reduction of outer segments of photoreceptors, reduction in the density of nuclear layers, and disintegration of ganglion cells. The dorsal portion of the retina experienced significantly greater deterioration than the ventral portion. Fish which had been subjected to 96 hours of continuous light displayed no ability to repair damage after 96 hours under 12L:12D.

Fluorescein angiography of the newborn rat. Implications in oxygen-induced retinopathy.

The current technique was developed to characterize the morphologic changes in the retinas of oxygen-reared rats, as an animal model of retinopathy of prematurity. Past studies have used ink perfusion to observe the retinal vasculature, but this method is static and requires the sacrifice of the subject. Fluorescein angiography, however, is dynamic and relatively noninvasive, and allows the survival of the animal for further study. The fundus camera cannot be used because the source of light that is focused in an annulus is too large for the pupil size of a young (approximately 14-day-old) rat. To overcome this, a Nikon inverted microscope (Diaphot-TMD) was used. Using the proper exciting and barrier filters for fluorescene, a photographic sequence was made by rapidly focusing to the plane of the retinal vessels. To our knowledge, similar photographs have not been previously published. This technique was used in newborn pigmented ratlings that were 1) exposed to 80% oxygen for the first 14 days of life; 2) exposed to 80% oxygen for the first 21 days of life; or 3) exposed for the first 14 days followed by 7 days in room air. Age-matched controls were raised simultaneously in room air and evaluated with the same technique. Differences were observed between treatments in the amount of retinal capillary loss, and in the tortuosity and diameter of the major retinal vessels. The hyaloid system also varied between treatment groups. Oxygen-exposed rats showed a persistence of the hyaloid vessels that was particularly prominent in the group returned to room air before analysis. Comparisons are made to past results obtained with other histologic techniques.

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.

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:

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.

Oxygen-induced retinopathy in the rat. Vitamins C and E as potential therapies.

Oxygen-induced retinopathy (OIR) was produced by subjecting newborn albino rats to a 60% oxygen atmosphere for 14 days before they were killed and retinal analysis was done. The extent of OIR was measured by estimating the severity of retinal vasoobliteration in ink-perfused flat-mounted retinas. This was done with the aid of a digitizing camera and an image-analysis system designed to create binary images of the retinal blood vessels. Retinal levels of several antioxidant molecules also were measured. Alpha-tocopherol and ascorbic acid were reduced in oxygen-exposed rats by 34% and 20%, respectively, compared with room air-raised control animals. Retinal glutathione reductase, S-transferase, and peroxidase showed no differences between oxygen-treated and -untreated rats. Attempts to increase the newborn rats' retinal ascorbic acid by administering daily subcutaneous injections (5 g/kg body weight) to the mother rats were unsuccessful. However, the level of retinal alpha-tocopherol of newborn rats could be altered by dietary manipulation of the mothers. The mothers were fed diets containing either 1 g alpha-tocopherol acetate/kg food or none, starting 21-25 days before the birth of their litters and lasting throughout the exposure period. This treatment resulted in three- to fourfold differences in the retinal alpha-tocopherol levels of the pups. The combination of dietary and oxygen treatments also resulted in significant differences in retinal glutathione peroxidase activity, with the vitamin E-deprived, oxygen-exposed group having highest levels. Newborn rats both supplemented with and deprived of alpha-tocopherol had less vasoobliteration than did those nursed by mothers fed rat chow.

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.

Effects of oxygen rearing on the electroretinogram and GFA-protein in the rat.

Albino rats were maintained in 60% oxygen from birth through 14 days of age. Control rats were simultaneously maintained in room air. At the end of the exposure period some animals from both groups were prepared for electroretinography and, following this analysis, were sacrificed for histological scrutiny of retinal vasculature. The rest of the rats were removed to room air and allowed to remain for various periods of time before electroretinography and sacrifice. Some of the retinas from the latter group were analyzed for the presence of glial fibrillary acidic protein (GFAP) by immunocytochemistry. Oxygen exposed rats had a permanent reduction of the b-wave of the electroretinogram (ERG). The a-waves of the two groups were indistinguishable. Control rats showed GFAP reactivity in the retinal astrocytes at all times. The oxygen-treated rats showed positive GFAP-staining in astrocytes at all times and in Müller cells from 2 weeks post-treatment through 8 weeks post-treatment, the last time point. Immunoblot analysis confirmed that the anti-GFAP reacted with a protein having a molecular weight and solubility characteristics like those of GFAP. These results indicate that Müller cells produce GFAP in response to oxygen-rearing in newborn rats and that this production occurs in the absence of any detectable neuronal cell death.

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.

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.

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.

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.

A new microspectrophotometric method for measuring absorbance of rat photoreceptors.

A method has been developed for obtaining visual pigment absorbance spectra from fixed, frozen sections of the albino rat retina. The retinas are transversely sectioned at various thicknesses on a cryo-microtome and the resulting slices are examined with a photon-counting microspectrophotometer. Problems caused by the limited pigment content of small receptors are reduced and measurements can be made at well-specified locations along the retinal section. This method is both precise and accurate and permits comparisons across sections and across animals.

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.

Fluorescein angiography as a means of assessing retinal vascular pathology in oxygen-exposed newborn rats.

Recently a new technique was reported in which examination of the retina of oxygen reared newborn rats was made using indirect fluorescence microscopy (Invest. Ophthal. Vis. Sci. 31:810). We have now improved this technique and systematically employed it as a means of studying oxygen-induced retinopathy in the newborn rat as an animal model for retinopathy of prematurity (ROP). Immediately after birth, litters of pigmented rats were exposed to an elevated oxygen atmosphere that varied in a cyclic fashion between 40% and 80% every 12 hours for 14 days. Rats raised simultaneously in room air served as controls. At the end of the treatment period, rats were deeply anesthetized, their pupils dilated, and 100 microliters of 3% sodium fluorescein was administered via tail venipuncture. After angiography, rats were awakened and returned to their mothers in room air for future assessment. Angiography was performed again at 4, 7, 38 or 56 days after oxygen treatment. Our results suggest that retinal pathology in the rat model parallels that of the human infant with ROP with respect to loss of patent retinal blood vessels in oxygen and subsequent abnormal vasoproliferation upon return to room air. Angiograms performed on room air-raised control rats at 14 days of age demonstrated our ability to resolve the smallest retinal capillaries. Angiograms conducted at the same age in oxygen-exposed rats revealed extensive areas of retinal non-perfusion and substantial arterio-venous shunting between adjacent primary vessels. If rats were maintained in room air for 4 or 7 days following oxygen exposure, a variety of pathologic signs became obvious including: 1) tortuosity of primary vessels mimicking human "plus disease", 2) frequent abnormal budding of tertiary vessels and capillaries, 3) retinal fluorescein leaks indicative of hemorrhages and 4) abnormal capillary tufts not evident at the time of removal from oxygen. By 38 and 56 days post-exposure, all four of these phenomena were still observed. Many of these pathologic phenomena could not have been observed with conventional methods currently used for retinal vascular assessment in small animals.

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 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.