apyrase and Retinopathy-of-Prematurity

Caffeine or ketorolac decrease the risk of retinopathy of prematurity and may act synergistically to improve beneficial effect. Combination of caffeine (Caff) and ketorolac (Keto) to prevent oxygen-induced retinopathy was studied.. Newborn rats exposed to room air (RA) or intermittent hypoxia (IH) consisting of 12% O2 during hyperoxia (50% O2) from birth (P0) had single daily IP injections of Caff from P0-P13 or saline; and/or ocular Keto (Acuvail, 0.45% ophthalmic solution) administered subcutaneously over the eyes from P5-P7. Pups were studied at P14 or placed in RA for recovery from IH (IHR) until P21. Eyes were examined for neovascularization, histopathology, growth factors, and VEGF-signaling genes.. Severe retinal damage noted during IHR in the untreated groups evidenced by hemorrhage, neovascularization, and oxygen-induced retinopathy (OIR) pathologies were prevented with Keto/Caff treatment. Keto and/or Caff treatment in IH also promoted retinal neural development evidenced by eye opening (92%, P < 0.001 vs. 31% in the placebo-treated IH group). No corneal pathologies were noted with Keto.. Caff or Keto given individually reduced retinal neovascularization, but the two drugs given together prevented severe OIR.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Apyrase; Arteries; Body Weight; Caffeine; Central Nervous System Stimulants; Choroid; Citrates; Dinoprost; Drug Synergism; Female; Hemorrhage; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Ketorolac; Neovascularization, Pathologic; Oxygen; Rats; Rats, Sprague-Dawley; Retina; Retinopathy of Prematurity; Signal Transduction; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

CCN1 (also called Cyr 61) is an extracellular matrix signaling molecule that has been implicated in neovascularization through its interactions with several endothelial integrin receptors. The roles of vascular endothelial growth factor (VEGF) in angiogenesis are well described. The aim of this study was to investigate the signal transduction mechanism of CCN1-PI3K/Akt-VEGF in retinopathy of prematurity (ROP), and the effects of CCN1 knockdown on ROP.. The oxygen-induced retinopathy (OIR) model was established in C57BL/6J mice exposed to a high concentration of oxygen. Retinas were obtained from the normoxia, OIR, OIR control (treated with scramble siRNA) and OIR treated (with CCN1 siRNA) groups. Retinal neovascularization (RNV) was qualitatively analyzed with ADPase staining and quantitatively analyzed by counting neovascular endothelial cell nuclei at postnatal day 17 when RNV reached a peak. mRNA level and protein expression of CCN1, p-Akt, and VEGF were measured by real-time PCR and Western blotting, and located with immunohistochemistry.. CCN1 depletion resulted in less neovascularization clock hour scores in the number of preretinal neovascular cells compared with the OIR treated group (1.28±0.83 versus 4.80±0.82; and 7.12±2.50 versus 23.25±2.35, respectively, both P<0.05). Furthermore, CCN1, p-Akt and VEGF mRNA, and protein were significantly expressed in the retina of the OIR and OIR control groups. Intravitreal injection of CCN1 siRNA significantly reduced PI3K/Akt-VEGF pathway expression of the OIR mouse model (all P<0.05). CCN1 siRNA significantly enhanced the avascular area and avascular diameter of OIR model (P<0.05). CCN1 siRNA decreased the levels of IL-1β, IL-6, and TNF-α significantly compared to the OIR group (P<0.05).. These results suggest that CCN1 plays an important role in RNV via the PI3K/Akt-VEGF signaling pathway. CCN1 may be a potential target for the prevention and treatment of ROP.

Topics: Animals; Animals, Newborn; Apyrase; Cysteine-Rich Protein 61; Cytokines; Endothelial Cells; Female; Gene Knockdown Techniques; Male; Mice; Mice, Inbred C57BL; Oncogene Protein v-akt; Oxygen; Phosphatidylinositol 3-Kinases; Retinal Neovascularization; Retinopathy of Prematurity; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

The adenosine A(2A) receptor (A(2A)R) modulates normal vascularization and pathologic angiogenesis in many tissues and may contribute to the pathogenesis of retinopathy of prematurity (ROP) characterized by abnormal retinal vascularization in surviving premature infants. Here, the authors studied the effects of the genetic inactivation of A(2A)R on normal retinal vascularization and the development of pathologic angiogenesis in oxygen-induced retinopathy (OIR), an animal model of ROP.. After exposure to 75% oxygen for 5 days (postnatal day [P] 7-P12) and subsequently to room air for the next 9 days (P13-P21), we evaluated retinal vascular morphology by ADPase staining in retinal whole mounts, retinal neovascularization response by histochemistry in serial retinal sections, and retinal VEGF gene expression by real-time PCR analysis in A(2A)R knockout (KO) mice and their wild-type (WT) littermates.. At P17, A(2A)R KO mice displayed attenuated OIR compared with WT littermates, as evidenced by reduced vaso-obliteration and areas of nonperfusion in the center of the retina, reduced pathologic angiogenesis as evident by decreased non-ganglion cells and neovascular nuclei, and inhibited hypoxia-induced retinal VEGF gene expression. Notably, the attenuation of pathologic angiogenesis by A(2A)R inactivation was selective for OIR because it did not affect normal retinal vascularization during postnatal development.. These findings provide the first evidence that A(2A)R is critical for the development of OIR and suggest a novel therapeutic approach of A(2A)R inactivation for ROP by selectively targeting pathologic but not developmental angiogenesis in the retina.

Topics: Animals; Animals, Newborn; Apyrase; Disease Models, Animal; Gene Silencing; Humans; Immunoenzyme Techniques; Infant, Newborn; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Physiologic; Oxygen; Polymerase Chain Reaction; Proliferating Cell Nuclear Antigen; Receptor, Adenosine A2A; Retina; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Vascular Endothelial Growth Factor A

The efficacy of three matrix metalloproteinase (MMP) inhibitors with various selectivities (Ro-31-9790, AG3340, and DPC-A37668) was investigated in a rat model of retinopathy of prematurity, to examine the roles of MMP-2 and -9 in retinal neovascularization. The susceptibilities of MMP-2(-/-) and -9(-/-) mice to preretinal neovascularization were investigated in a mouse model of oxygen-induced retinopathy.. Sprague-Dawley newborn rats were exposed to alternating episodes of 50% and 10% oxygen (variable oxygen exposure) to induce retinal neovascularization. Three MMP inhibitors with various selectivity profiles were administered to variable oxygen-exposed rats via local or systemic routes. Antineovascular efficacy was determined in drug-treated versus vehicle-treated rat pups by computerized imaging of adenosine diphosphatase (ADPase)-stained retinal flatmounts. Wild-type C57BL/6J and isogenic MMP-2(-/-) and -9(-/-) mice were exposed to 75% oxygen followed by normoxia. The mice were killed immediately before or after the normoxic exposure, and eyes were either harvested for retinal dissection and flatmounting or were paraffin embedded and sectioned. Retinal vascular area and retinal neovascularization were assessed by adenosine diphosphatase staining of retinal flatmounts and by counting preretinal nuclei of hematoxylin and eosin-stained retinal sections, respectively.. Ro-31-9790, AG3340, and DPC-A37668 had no effect on normal development of the rat retinal vasculature, regardless of dose or route of administration. Intravitreal injection of Ro-31-9790 (broad-spectrum) immediately after variable-oxygen exposure and 2 days after exposure resulted in 78% and 82% inhibition of retinal neovascularization, respectively. AG3340 (MMP-2- and -9-selective inhibitor) and DPC-A37668 (MMP-2-selective inhibitor) resulted in 65% and 52% inhibition, respectively, when administered by intravitreal injection immediately after variable-oxygen exposure. Intraperitoneal injection of 5, 15, and 50 mg/mL AG3340 or DPC-A37668 for 6 days after variable oxygen exposure resulted in 22% to 39% and 0% to 31% inhibition of neovascularization, respectively. AG3340 and DPC-A37668 administered by oral gavage at doses of 3, 10, or 30 mg/mL provided up to 42% and 86% inhibition of neovascularization, respectively. The average vascular areas of retinas from MMP-2(-/-) or -9(-/-) mice at postnatal day 12 were not significantly different from the wild-type control. There was a 75% (P < 0.001) and 44% (P < 0.01) reduction in preretinal neovascularization in oxygen-exposed MMP-2(-/-) and -9(-/-) mice at postnatal day 19, respectively, compared with wild-type control mice.. The results of this study suggest that MMP-2 plays a predominant role in retinal angiogenesis in both the mouse and rat models of oxygen-induced retinopathy. Furthermore, MMP-2 inhibition may be a viable therapeutic approach for ocular diseases characterized by retinal neovascularization.

Topics: Animals; Animals, Newborn; Apyrase; Disease Models, Animal; Enzyme Inhibitors; Gene Silencing; Humans; Hydroxamic Acids; Infant, Newborn; Injections; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Organic Chemicals; Oxygen; Pyridines; Rats; Rats, Sprague-Dawley; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Vitreous Body

To test the hypothesis that in experimental retinopathy of prematurity (ROP), retinal neovascularization (NV) and vessel tortuosity have distinct spatial and temporal links with receptor and postreceptor ion demand.. Newborn rats were raised in either room air (controls) or variable oxygen (50%/10% [50/10]). After 14 days, 50/10 rats were recovered in room air until postnatal day (P) 19 or P22. Peripheral retinal NV severity and incidence and panretinal arteriole and venule tortuosity indexes (TI(a), TI(v)) were measured from ADPase-stained retinal wholemounts. Intraretinal ion demand and retinal thickness were measured from high-resolution manganese-enhanced MRI (MEMRI). In separate experiments, intraretinal manganese uptake was also measured in adult rats pretreated with diltiazem, a Ca(2+) channel antagonist.. In 50/10 rats, peripheral retinal NV severity was significantly greater than in controls at P19 and was decreased by P22. Panretinal TI(a) and TI(v) were increased over control values at P19, but only TI(v) decreased by P22. Unlike control retinas at P19 that had a centroperipheral total retinal thickness gradient, 50/10 retinas had similar central and peripheral total retinal thickness. The 50/10 group also demonstrated a correlation between peripheral retinal NV and TI(a) and TI(v). Peripheral intraretinal uptake of manganese was significantly supernormal at P19 and decreased by P22. Increased peripheral intraretinal retinal manganese uptake was associated with peripheral NV severity and panretinal TI(a). In contrast, ion demand of central postreceptor, but not receptor, retina was significantly associated with peripheral NV severity and panretinal TI(a). Panretinal TI(v) was not correlated with intraretinal ion demand in any case. In adult rats, diltiazem suppressed (P < 0.05) intraretinal manganese uptake.. The present data raise the possibility that altered retinal layer-specific ion demand causes retinal circulation abnormalities in experimental ROP.

Topics: Animals; Animals, Newborn; Apyrase; Calcium Channel Blockers; Diltiazem; Disease Models, Animal; Female; Humans; Infant, Newborn; Magnetic Resonance Imaging; Manganese; Photoreceptor Cells, Vertebrate; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinopathy of Prematurity

To test the hypothesis that retinal hypoxia is present during vascular development in normal rat pups and in a newborn rat model of retinopathy of prematurity (ROP).. Preretinal vitreous PO(2) measurements were made during room air breathing using (19)F magnetic resonance spectroscopy (MRS) and a perfluoro-15-crown-5-ether droplet in normal adult and newborn (postnatal day [P]1-P20) rats, and in newborn rats exposed first to 14 days of variable oxygen (before NV) and six additional days in room air after variable oxygen exposure (during NV). After each experiment, blood gas values were measured, and retinas were isolated. Retinas were adenosine diphosphatase (ADPase) stained, and flatmounted to determine peripheral avascular extent and NV incidence and severity.. In the vascularized rat retina, no significant difference (P > 0.05) was found between the droplet-derived preretinal vitreous oxygen tension (24 +/- 2 mm Hg, mean +/- SEM, n = 18) and previously reported electrode-measured oxygen tension (22 +/- 1 mm Hg). Only during normal retinal vessel growth (P1-P10) and before the appearance of NV was evidence for retinal hypoxia found at the border of the vascular and avascular retina. However, the mean PO(2) (range, 24-28 mm Hg) over the vascular retina was not different (P > 0.05) between any of the newborn rat groups in this study.. (19)F MRS of a perfluorocarbon droplet provides an accurate measure of preretinal vitreous oxygen tension in rats. These data support an important role of physiologic hypoxia in normal retinal circulatory development and raises the possibility that, in experimental ROP, retinal hypoxia is a necessary but not sufficient condition for the development of retinal NV.

Topics: Animals; Animals, Newborn; Apyrase; Crown Ethers; Disease Models, Animal; Ethers, Cyclic; Female; Humans; Hypoxia; Infant, Newborn; Magnetic Resonance Imaging; Male; Oxygen; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity

Vascular endothelial cell growth factor (VEGF) has been implicated in vascular development and in proliferative retinopathies. The goal of this study was to examine the immunohistochemical localization and relative levels of VEGF receptor-2 (KDR) in canine retina during postnatal vasculogenesis and during angiogenesis in oxygen-induced retinopathy (OIR) and to investigate the effects of neutralizing KDR on these processes.. Eyes from normal dogs ranging from 1 to 22 days of age and age-matched oxygen-treated animals were snap frozen for immunohistochemical analysis with antibodies against human KDR. To examine the effects of blocking KDR, 6-day-old air-reared control and oxygen-treated animals were surgically implanted with slow release polymer pellets containing control IgG or anti-KDR. Material eluted from pellets was assessed using a binding assay (measures binding to soluble KDR) to determine the kinetics of anti-KDR release and endothelial cell proliferation to measure bioactivity. Animals were killed at 22 days of age and tissues examined with adenosine diphosphatase (ADPase) histochemical staining of blood vessels.. KDR immunoreactivity was only weakly associated with developing retinal vessels and was not observed in angioblasts throughout normal postnatal development. Immunoreactivity was very strong in reforming retinal vessels and intravitreal neovascularization in oxygen-treated animals. Anti-KDR had no effect on vessel morphology or growth in air-reared control animals. In oxygen-treated animals, anti-KDR significantly inhibited revascularization of the retina (P = 0.005) and formation of intravitreal neovascularization compared with control IgG pellet eyes (P < 0.04).. KDR/Flk-1 was only weakly associated with normal developing primary retinal vessels but was strongly expressed by proliferating endothelial cells in reforming retinal vessels and intravitreal neovascularization after hyperoxic insult. Anti-KDR antibody delivered by slow-release pellets had no effect on normal vasculogenesis, but it inhibited the formation of intravitreal neovascularization and retinal vessel development in OIR. The study suggests that blocking KDR may be beneficial for treating pathologic angiogenesis in adult tissue.

Topics: Animals; Animals, Newborn; Antibodies, Blocking; Apyrase; Disease Models, Animal; Dogs; Endothelium, Vascular; Humans; Hyperoxia; Immunoenzyme Techniques; Immunoglobulin G; Infant, Newborn; Neovascularization, Physiologic; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Retina; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity

We have previously described a metabolic acidosis-induced retinopathy in the neonatal rat, similar to retinopathy of prematurity (ROP). We also have reported exacerbation of oxygen-induced retinopathy by postnatal growth retardation, produced by raising newborn rats in 'expanded' litters. In the present study, we investigated the effect of postnatal growth retardation on the incidence and severity of acidosis-induced retinopathy.. 100 newborn Sprague-Dawley rats were randomly assigned to two expanded litters of 25 pups each and five standard control litters of 10 pups each. All rats were gavaged with 10 mM/kg NH(4)Cl twice daily from days two to seven. Following five days of recovery, retinal vasculature was assessed using ADPase staining, light microscopy, and computer-assisted image analysis. The presence of neovascularization (NV), severity of NV (clock hours), and vascularized retinal areas, were evaluated in a masked manner.. NV occurred in 52% of rats in expanded litters versus 18% of rats in standard control litters (p = 0.005). Postnatal growth retardation of pups in expanded litters was confirmed by comparing total body weight of pups raised in expanded and standard control litters (10.8g vs 13.4g on day 8, p < 0.001; 20.8g vs 25.2g on day 13, p = 0.002).. Postnatal growth retardation increases the incidence of acidosis-induced retinopathy in the neonatal rat. Our study provides further evidence that postnatal growth retardation is a risk factor for preretinal neovascularization in immature retinae and is consistent with the clinical observation that the smallest and sickest premature infants are more likely to suffer from ROP.

Topics: Acidosis; Ammonium Chloride; Animals; Animals, Newborn; Apyrase; Blood Gas Analysis; Body Weight; Growth Disorders; Humans; Hydrogen-Ion Concentration; Image Processing, Computer-Assisted; Infant, Newborn; Litter Size; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Risk Factors

The method of counting cell nuclei above the internal limiting membrane in histologic sections is considered the standard when quantifying neovascularization (NV) in rodent oxygen-induced retinopathy (OIR). An alternative, more rapid method of counting clock hours in flatmounted adenosine diphosphatase (ADPase)-stained rat retinas is analogous to clinically scoring retinopathy of prematurity (ROP). In the present study, the validity of counting clock hours was evaluated by a direct comparison of these techniques. The intereye correlation of NV score and retinal vascular area were also studied.. Newborn Sprague-Dawley rats were exposed to cycles of O2 (80-10%) for 7 days, followed by 5 days of room air recovery. Preretinal NV was quantified by three masked observers counting clock hours in flatmounted ADPase-stained retinas of both eyes. Retinal vascular and total retinal areas were calculated using computer-assisted analysis. Representative retinas that had been scored positive (n = 10) and negative (n = 3) for NV and room air control retinas (n = 3) were embedded in paraffin. Each entire peripheral retinal quadrant was serially sectioned at 6 microm and stained with hematoxylin and eosin. Nuclei above the internal limiting membrane were then counted in a masked manner. The total number of nuclei counted per retina was defined as the nucleus count (704-938 sections per retina; 12,900 sections). Correlations were evaluated using Spearman rank coefficients.. The nucleus count was 0 to 44 in room air control retinas, 0 to 40 in negative OIR retinas, and 250 to 5634 in positive OIR retinas. The nucleus count was highly correlated with the clock hour score (r(s) = 0.95, P = 0.0001). For the paired retinas, there was a significant correlation between right and left eyes in the severity of NV (clock hours; r(s) = 0.76, P = 0.0001) and the ratio of retinal vascular area to total retinal area (r(s) = 0.81, P = 0.0001).. The more rapid method of counting clock hours in flatmounted ADPase-stained retinas is valid for quantifying NV in rat models of ROP. Incidence and severity of NV and vascularized areas were similar between left and right eyes, which permits the use of paired retinas for complementary research techniques.

Topics: Animals; Animals, Newborn; Apyrase; Cell Nucleus; Disease Models, Animal; Humans; Infant, Newborn; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinopathy of Prematurity

Carbon dioxide (CO2)-induced retinopathy (CDIR) in the neonatal rat, analogous to human retinopathy of prematurity (ROP), was previously described by our group. In this model, it is possible that CO2-associated acidosis provides a biochemical mechanism for CDIR. Therefore, the effect of pure metabolic acidosis on the developing retinal vasculature of the neonatal rat was investigated.. A preliminary study of arterial blood pH was performed to confirm acidosis in our model. In neonatal rats with preplaced left carotid artery catheters, acute blood gas samples were taken 1 to 24 hours after gavage with either NH4Cl 1 millimole/100 g body weight or saline. In the subsequent formal retinopathy study, 150 newborn Sprague-Dawley rats were raised in litters of 25 and randomly assigned to be gavaged twice daily with either NH4Cl 1 millimole/100 g body weight (n = 75) or saline (n = 75) from day 2 to day 7. After 5 days of recovery, rats were killed, and retinal vasculature was assessed using fluorescein perfusion and ADPase staining techniques.. In the preliminary pH study, the minimum pH after NH4Cl gavage was 7.10+/-0.10 at 3 hours (versus 7.37+/-0.03 in controls, mean +/- SD, P < 0.01). In the formal retinopathy study, preretinal neovascularization occurred in 36% of acidotic rats versus 5% of controls (P < 0.001). Acidotic rats showed growth retardation (final weight 16.5+/-3.0 g versus 20.2+/-2.6 g, P < 0.001). The ratio of vascularized to total retinal area was smaller in acidotic rats (94%+/-4% versus 96%+/-2%, P < 0.001).. Metabolic acidosis alone induces neovascularization similar to ROP in the neonatal rat. This suggests a possible biochemical mechanism by which high levels of CO2 induce neovascularization and supports the suggestion that acidosis may be an independent risk factor for ROP.

Topics: Acidosis; Ammonium Chloride; Animals; Animals, Newborn; Apyrase; Blood Gas Analysis; Fluorescein Angiography; Humans; Hydrogen-Ion Concentration; Infant, Newborn; Random Allocation; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Risk Factors

Hypercarbia has been suggested as a risk factor for retinopathy of prematurity (ROP). We investigated the effect of hypercarbia on the retinal vasculature of the neonatal rat to determine whether hypercarbia alone could induce preretinal neovascularization analogous to ROP.. In a preliminary blood gas study, 8-11-day-old rats were exposed to specific levels of inspired O2 and either 0.2% CO2 or 10% CO2. Arterial blood gases were obtained, and a level of inspired O2 was determined that, when combined with inspired 10% CO2, would produce a PaO2 equivalent to room air (pure hypercarbia). In the formal retinopathy study, 300 newborn rats raised in 12 expanded litters (n = 25 each) were exposed for 7 days to either room air, 10% CO2 in 21% O2 and nitrogen (high-inspired CO2 group) or 10% CO2 in 12.5% O2 and nitrogen (pure-hypercarbia group). Each type of exposure was followed by recovery in room air for 5 days. Animals were sacrificed on day 13 and retinae were analyzed, using fluorescein perfusion and ADPase staining techniques.. Neovascularization occurred in 19% of rats in the high-inspired CO2 group, and 14% of rats in the pure-hypercarbia group, compared to 0% of rats exposed to room air alone (p = 0.001, Chi square).. In the neonatal rat model, exposure to hypercarbia alone, followed by room air recovery, results in preretinal neovascularization similar to that seen in oxygen-induced retinopathy. Our results support the suggestion that hypercarbia may be a risk factor for retinopathy of prematurity.

Topics: Animals; Animals, Newborn; Apyrase; Blood Gas Analysis; Carbon Dioxide; Humans; Hypercapnia; Hyperoxia; Infant, Newborn; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Risk Factors

In previous studies the morphologic features of the acute vaso-obliterative and vasoproliferative stages of oxygen-induced retinopathy (OIR) were quantified and described in the dog model of retinopathy of prematurity (ROP). In the present study the sequelae of these events were examined using fluorescein angiography and histologic, enzyme, and immunohistochemical techniques.. Thirty newborn animals were exposed to 95% to 100% oxygen for 4 days and returned to room air until they were 22 to 45 days of age. Before death some animals were anesthetized, and fluorescein angiography was performed. Retina and vitreous from some animals were processed for adenosine diphosphatase (ADPase) flat-embedding. In other cases, eyes were prepared for full-thickness eyewall sectioning or frozen for histochemical analysis.. Fluorescein angiography, funduscopic examination, and ADPase preparations showed dilated and tortuous retinal vessels, pigmentary changes, incomplete vascularization of peripheral retina, vitreous hemorrhage, and persistence of massive intravitreal neovascularization. Full-thickness eyewall sections showed tractional retinal folds, tented intravitreal vascularized membranes, and vitreous synchysis. Immunohistochemical analysis showed inner retinal astrogliosis. Enzyme histochemistry showed high alpha glycerophosphate dehydrogenase activity in poorly differentiated neovascular formations and low activity in formations with mature pericytes and endothelial cells.. End-stage OIR in the neonatal dog shares many features with the chronic human disease. These results provide additional support for the use of this model in experimental studies of ROP.

Topics: Animals; Animals, Newborn; Apyrase; Astrocytes; Disease Models, Animal; Dogs; Fluorescein Angiography; Fundus Oculi; Glial Fibrillary Acidic Protein; Glycerolphosphate Dehydrogenase; Humans; Hyperoxia; Immunoenzyme Techniques; Infant, Newborn; Oxygen; Retina; Retinal Neovascularization; Retinopathy of Prematurity; Vitreous Hemorrhage; von Willebrand Factor

To quantify the acute constrictive response of developing retinal blood vessels to hyperoxia and to examine the vaso-obliterative phase of sustained oxygen breathing in the neonatal dog model of retinopathy of prematurity.. Seven littermates were used to examine the acute constrictive response of the developing retinal vessels to hyperoxia (30 minutes to 96 hours of 100% oxygen). ADPase retinal flatmounts were prepared, and morphometric measurements were made using computer-assisted analysis. Vaso-obliteration also was examined in three animals killed after prolonged exposure to hyperoxia (4 days of 100% oxygen) and in three room air controls using ADPase flat-embedded retinas and cross-sections. Choroids were processed for alkaline phosphatase flat-embedding.. After 1 hour of oxygen breathing, all vascular components showed a reduction in diameter: Arteries were reduced 27%, veins 18.3%, and capillaries 27.7%. Capillary constriction peaked by 24 hours (69.4% reduction), whereas arteries and veins continued to close. Although capillary diameters did not decrease significantly after 24 hours, the number of capillaries, as determined by percent vascular area calculations, continued to decrease in all areas through the additional 3 days of oxygen breathing. In contrast, after 4 days of hyperoxia the choriocapillaris lumenal diameters and percent vascular area did not vary significantly from controls. Analysis of sections taken through various retinal regions of these animals revealed significant decreases (40%) in the volume of the extracellular spaces available for blood vessel formation. Hyperoxia also reduced in a 55.6% decrease in the total number of cells (endothelial cells, ablumenal cells, perivascular cells) within the inner retina; however, there was no significant difference in ganglion cell counts in the two groups.. This study demonstrates that the pattern and severity of the reaction of developing retinal vessels to hyperoxia in the newborn dog is similar to that described for the kitten and the premature human. This response is unlike that exhibited by the newborn rat or mouse.

Topics: Animals; Apyrase; Cell Count; Choroid; Disease Models, Animal; Dogs; Humans; Hyperoxia; Infant, Newborn; Mice; Oxygen; Rats; Retinal Artery Occlusion; Retinal Vein Occlusion; Retinal Vessels; Retinopathy of Prematurity

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.. 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.. 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.. 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.

Topics: Animals; Animals, Newborn; Apyrase; Capillaries; Disease Models, Animal; Histocytochemistry; Humans; Image Processing, Computer-Assisted; Infant, Newborn; Oxygen; Rats; Rats, Sprague-Dawley; Retinal Vessels; Retinopathy of Prematurity

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.. 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.. 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.. 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.

Topics: Animals; Animals, Newborn; Apyrase; Disease Models, Animal; Female; Humans; Image Processing, Computer-Assisted; Incidence; Infant, Newborn; Male; Oxygen; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity