The Inhibition Effect Of Pigment Epithelium-derived Factor On Retinal Neovascularization Of Oxygen-induced Rat Models

BackgroundROP is proliferative disease of the retinal vasculature infants that may cause severe visual loss, and it is a major cause of blindness in newborn, The disease is characterized by proliferation of abnormal fibrovascular tissue at the border of vascularised and non-vascularised retina. Retinopathy of prematurity (ROP) is a potenitially blinding disease affecting premature infants. The immature neurovascular tissues of the visual system, the retina and brain, are particularly susceptible to injury. ROP is characterized by pathological ocular angiogenesis or retinal neovasculrization (NV).This NV leads to traction retinal or further proliferation of fibrovascular tissue on the retinal surface, resulting in formation of a retrolental fibrovascular membrane, total retinal detachment, intravitreal hemorrhages and subsequent vision loss. The approved treatments consist in photocoagulation surgery, laser photocoagulation or cryotherapy. However, the visual outcomes after treatment often are not satisfactory. While guidelines for the management and treatment of ROP have recently been refined, it is still difficult to identify those infants in whom disease is more likely to progress. Preventive therapy for ROP is sorely needed.Abnormal development of the retinal vasculature is at the heart of this disease. Vascular endothelial growth factor(VEGF) has emerged as one of the most important in the development of NV. PEDF is a member of the serine protease inhibitor (serpin) family, although it does not have protease inhibitory activity. Pigment epithelium-derived factor(PEDF) is the most potent natural inhibitor of angiogenesis. PEDF has emerged as a potentially important endogenous inhibitor of ocular neovascularization. PEDF is a multifunctional serpin protein. It is PEDF also has neuroprotective activity and has been demonstrated to protect photoreceptors from degeneration. PEDF has been found in the vitreous and cornea, and it has been found to be downregulated by hypoxia in cultured cells. Its presence in the vitreous and its antiangiogenic activity suggest a possible role for PEDF in the regulation of retinal neovascularization. PEDF strongly inhibited VEGF-induced migration and proliferation of retinal endothelial cells.Therefore, our experiment used oxygen concentration variation to establish a suitable oxygen-induced retinopathy rat model for imitating human ROP. We analysed the relationship between VEGF and retinal neovascularization, We observed the effects of PEDF on OIR in rats with subconjunctival or intravitreal injection. We strived a novel, safer and more effective therapeutic approaches for the treatment of pathologic neovascular conditions.BackgroundRetinopathy of prematurity (ROP) is a causing-blindness eye disease Oxygen in premature retinal neovascularization plays an important role, but its scope of oxygen and the specific mechanism of retinopathy is not clear ObjectiveTo study the effects of different concentrations of oxygen on neovascularization in oxygen-induced retinopathy (OIR) of newborn rats and provide experimental basis for clinical oxygen therapy; To determine the roles of vascular endothelial growth factor (VEGF)in vascularization of the developing retina.Method:Forty Sprague-Dawley rats were included in the study. They were divided into four groups:Air group (10rats) was was raised in air room. Fluctation group1、2、3(10rats) was exposed to cycles of50%～20%,40%～10%,50%～10%for14days, respectively. In each case, oxygen was alternated between the two exposure concentrations every24hours. The rats were removed to room air for4days before the severity of abnormal neovascularization were measured. Retinas were dissected and stained by adenosine-diphosphatase(ADPase) histochemistry for assessmem of intraretinal vascular development and preretinal angiogenic vessel growth. The proliferative neovascularization response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. The total number of nuclei counted per retina was defined as the nucleus count. Nuclei above the internal limiting membrane were counted. Protein expressions of VEGF were studied by western-blot.Statistical analysis:The analysis was performed using SPSS13.0software package. The data was represented as the mean±tandard deviation(x±s), Comparisons of means among group were performed using one-way analysis of variance(ANOVA). If Variance were homogenous among groups, the LSD method was used for multiple comparisons, otherwise Dunnett C method was used, P<0.05were considered to be statistically significant.Result Retina flat-mounts (ADP-ase staining):The retinal vascularization had completed in normal air group, The main buanches of vessels in optic idsc are well-developed in normal air group. Retina vascular development is normal basically;Retina flat-mounts presented increasing neovascular tufts,plenty of newly born vascular plexus and disorderly arranged vessel structures in Fluctation group2and Fluctation group3.The clock number of these four groups were0,0.17±0.41,4.8±1.2,7.8±1.2,respectively. the difference was significant (F=120.21, p=0.00). The difference was statistically significant between Fluctation group2and Fluctation group3(P=0.00).HE stain results:The number of vascular nuclei extending from the internal limiting membrane into the vitreous were0.6±0.47,0.87±0.65,14.28±3.8322.52±4.02, respectively. The difference was significant (F=875.366, p=0.00). The differences in the VEGF-expression among three groups were significant (P=0.00).Conclusion1.50%～10%pattern is suitable for oxygen-induced retinopathy rat model to imitate human ROP; This animal model(50%～10%pattern) of OIR is reproducible. Because of its quantifiable characteristic, it is useful for the study of pathogenesis of retinal neovascularization and theraputic intervention for ROP.2. Changeable Oxygen concentrationa could trigger a subsequent neovascularization;△FiO2were positively correlated with the retinopathy;3. Hypoxia may be more important for retinal neovascularization on the occurrence of retinopathy than Hyperoxia;4. The over-expression of VEGF plays a important role during the formation of neovascularization. ObjectiveTo observe the inhibitive effect of intravitreal or Subconjunctival injecting PEDF for retinal neovascularization in Oxygen-induced animal models.MethodMouse models of oxygen-induced retinopathy of prematurity were established.48newborn rats were randomly diveded into6groups (A: Air control group; B:Hyperoxygen control guoup; C:Hyperoxygen+intravitreal injection of2ugPEDFgroup; D:Hyperoxygen+injection of2ugPEDFgroup; E:Hyperoxygen+injection of4ugPEDFgroup; F:Hyperoxygen+injection of8ugPEDFgroup). When the rats were taken off the high oxygen, the PBS and corresponding dosage of PEDF were intravitreal or Subconjunctival injected in group C, D, E and F, respectively. ADP-ase stained retina flat-mounts was used to evaluate the retinal vascular modality and development, Eyeball pathological section was used to count the pre-retinal endothelial nucleus in retinal new vessel. Retinal PEDF expression were detected by western-blot.Statistical analysis:The analysis was performed using SPSS13.0software package. The data was represented as the mean±standard deviation(x±s), Comparisons of means among group were performed using one-way analysis of variance(ANOVA). If Variance were homogenous among groups, the Bonferroni method was used for multiple comparisons, otherwise Dunnett C method was used, P<0.05were considered to be statistically significant.ResultsPEDF were achieved in retinal after a periocular injection. Retinal flat-mounts: A group:The retina vessels of the normal had a fine radial branching pattern that extend from the optic nerve to the periphery; B group:There were more retinal neovascularization compared with A group.The density and shape of retinal neovascularization were disordered; Comparing with B group, retinal neovascularization decreased obviously in C group while D, E, F group decreased mildly. HE staining:A group:Retinal internal limiting membrane was smooth, and occasionally the endothelial cells break through the inner retinal. B group was significantly more than group A(p<0.05). C group was significantly less than the B group (p<0.05), D, E, F group had significant difference between group C (all p <0.05).Conclusion1. PEDF are able to penetrate the sclera into retinal after a periocular injection, however, compared with intravitreal injection, the effect reduced significantly.2. Intravitreal injection of PEDF can arrest retinal neovascularization in SD rat models;3. How to reach the retina effectively requires further study by subconjunctival injection.