| Objective: Choroidal neovascularization(CNV) is one of the important manifestation patterns of intraocular neovascular formation and is the basic pathological change of various intraocular diseases. However, due to the awareness of CNV started relatively late, there are still many pressing issues in the pathogenesis, clinical diagnosis, treatment and other aspects to be addressed.So it is one of the important topics in ophthalmology. During the pathogenesis of CNV, retinal pigment epithelium(RPE) cells play important regulatory roles; and choriocapillary endothelial cells are the target cells. Under normal conditions, there is an important barrier between the above two key types of cells, known as the Bruch’s membrane. Once the Bruch’s membrane is impaired, various stimulating factors can act on the choriocapillary endothelial cells directly or indirectly through the RPE cells, triggering CNV. It is generally believed that CNV is related to the changes of RPE-Bruch’s membrane-choriocapillary complex. The latest studies found that Rap1 A plays an important role in maintaining the integrity of RPE barrier, while Rap1 B plays an important role in controlling and preventing the choroidal vascular endothelial cells from penetrating through the RPE and invading into the neurosensory retina. This study focused on strengthening the integrity of RPE barrier as the breakthrough point,and established the laser-induced rat CNV model, to observe the expressions of Rap1, GTP-Rap1, VEGF and β-catenin in experimental CNV from the perspectives of histopathology and molecular pathology,in order to provide a research foundation for Rap1-targeted consolidation of RPE barrier integrity in the future.Methods:1 An experimental study on the expression of Rap1 in choroidal neovascularization 1.1 Thirty 8-10 week healthy BN rats, male or female, without eye diseases both by direct ophthalmoscope and slit lamp examination,were randomly divided into blank control group, 3 days after laser group,7 days after laser group,14 days after laser,21 days after laser group, 28 days after laser group, 5 rats each group. 1.2 Laer-induced CNV model:Krypton laser(wavelength 647 nm,spot diameter of 200μm,power of 260 m W,exposure time 0.05s) around the optic disc photocoagulation 9-10 points,see any air bubbles produced by the breakdown Bruch membrane. 1.3 Fundus fluorescein angiography(FFA) was performed on days of laser 3,7, 14, 21,28,to observe CNV formation 1.4 The rats were killed after angiography,removal of the eye, posterior segment to make paraffin sections, selected a clear laser spot, HE staining.Then randomly selected the paraffin sections on 3,7,14,21,28 day after photocoagulation,make immunohistochemical staining,detect the expression of Rap1. 1.5 Image analysis and statistical processingSPSS19.0 software was used to analyze the positive staining rate obtained at each time point under a microscope to examine the difference of Rap1 expression in normal tissues and in photocoagulated tissues. 2 An experimental study on the expression of Rap1,GTP-Rap1,VEGF and β-catenin in choroidal neovascularization 2.1 Thirty 8-10 week healthy BN rats, male or female, without eye diseases both by direct ophthalmoscope and slit lamp examination,were randomly divided into blank control group, 3 days after laser group,7 days after laser group,14 days after laser,21 days after laser group, 28 days after laser group, 5 rats each group.Laer-induced CNV model:Krypton laser(wavelength 647 nm,spot diameter of 200μm,power of 260 m W,exposure time 0.05s) around the optic disc photocoagulation 9-10 points,see any air bubbles produced by the breakdown Bruch membrane. 2.2 Two rats were randomly selected from each group(n=4),the protein levels were determined with Western Blot to analyze the expressions of Rap1, GTP-Rap1, VEGF and β-catenin when. 2.3 The remaining rats in each group were selected(n= 6), the Real-Time PCR technique was used to determine the changes in m RNA transcription levels of Rap1, GTP-Rap1, VEGF and β-catenin genes in each group of experimental CNV 2.4 All the data was statistically analyzed using SPSS19.0 softwareResults: 1 An experimental study on the expression of Rap1 in choroidal neovascularization 1.1 FFA examination: Large areas of disciform fluorescein leakage were observed in 14 d after laser treatment; the number of photocoagulation plaques with fluorescein leakage peaked in 21 d after laser treatment.1.2 Light microscopy: Early-stage CNV in photocoagulation area was found in 7d after laser treatment; significant fibrovascular proliferation in CNV and extensive neovascularization were observed in 21 d after laser treatment. 1.3 There was no significant change in Rap1 expression in 3~28d after laser treatment when compared with the blank control group 2 An experimental study on the expression of Rap1,GTP-Rap1,VEGF and β-catenin in choroidal neovascularization 2.1 Western Blot results: There was no significant change in Rap1 expression at different time points after laser treatment when compared with blank control group,GTP-Rap1 expression significantly decreased at different time after laser treatment points and showed a downward trend when compared with blank control group,VEGF expression significantly increased in 7d and 14 d and peaked in 21 d after laser treatment;β-catenin expression at different time points after laser treatment showed an upward trend. 2.2 Real-Time PCR results: There was no change in Rap1 expression in each group and blank control group after laser treatment. GTP-Rap1, VEGF and β-catenin gene data in each group were all consistent with homogeneity of variance and normal distribution. There were statistically significant differences in GTP-Rap1 and VEGF in 7d, 14 d, 21 d and 28 d after photocoagulation between each group and blank control group. There were statistically significant differences in β-catenin expression at different time points after laser treatment when compared with blank control group.Conclusions:1. Rap1 expression in normal tissues and experimental CNV essentially remained unchanged,and then GTP- Rap1 made on RPE barrier adjustment.2. GTP-Rap1 expression decreased significantly after laser treatment when compared with normal rats, which was correlated with the formation and development of CNV.3 Since GTP-Rap1 was negatively correlated with VEGF and β-catenin expression, we speculated that the activation of Rap1 played an important role in maintaining the integrity of RPE barrier and inhibiting migration of choroidal vascular endothelial cells. |
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