Investigation Of Somatostatin Analogue Octreotide's Effect On The Experimental Choroidal Neovascularization

Objective: To observe the effect of somatostatin analogue octreotide on IGF-1, VEGF, and ultrastructural findings of RPE cells in the experimental choroidal neovascularization (CNV) of mice, investigating octreotide as an inhibitor on the formation of CNV.Methods: Models of laser-induced CNV: C57BL/6 mice were anesthetized with ketamine(50mg/Kg), and the pupils were dilated with a single drop of 0.5% tropicamide and 0.5% phenylephrine hydrochloride. Diode laser (Viridis-Lite, BVI, France) photocoagulation was used to generate ten spots in each eye surrounding the optic nerve through three-mirror lens. Conditions: wavelength 532nm, spot size 50μm, exposure time 0.1 seconds, power 250 mW. A bubble formed at a laser spot indicated the rupture of Bruch's membrane.We chose post-photocoagulation 7d, 14d, 21d and 28d as observed time point. In all 78 mice, 2 mice as normal control, each in one observed point as diseased control, 72 mice treated with octreotide were divided into 2 groups: group PO was treated as soon as finished laser photocoagulation, group P7 was treated 7 days after photocoagulation. Each groups had three sub-groups: simply treated with subcutaneous injection of octreotide, with retrobulbar injection, or two methods combined. The dose of octreotide was 50 μ g/kg, qd×14d. At each observed point, mice were sacrificed, the eyes were removed, the sclera-choroid-retina complex was flatmounted, stained with haematoxylin and eosin (HE), polyclonal IGF-1 antibody or polyclonal VEGF antibody for immunohistochemistry, observed by a light microscope. The image analysis system was used to analyze the flatmounts, measurethe area of choroidal neovascular membranes (CNVMs), compare the changes ofIGF-1 positive area or VEGF positive area in CNVMs.Reverse transcription poly chain reaction (RT-PCR) was used to detect the changes ofIGF-1 or VEGF mRNA by different medications at different observed point,analysising the expression intensity and relative coefficient.Western blotting was used to detect the expression of IGF-1 protein or VEGF proteinin the sclera-choroid-retina complex. The ultrastructural changes of RPE cells wereobserved at post-photocoagulation 21d in diseased control and group P7 treated withtwo methods combined.All data were analyzed with SPSS 12.0, using single factor analysis of variance and qanalysis (Newman-Keuls), chi-square test for two means. P<0.05, difference wassignificant; P<0.01, difference was extremely significant.Results: Immunohistochemical visualization showed that the positive expression ofIGF-1 in CNVMs could not be inhibited by subcutaneous injection of octreotide in theearly stage in group PO. In the late stage, it significantly decreased. In this group,retrobulbar injection or two methods combined were both effective. In group P7,subcutaneous injection of octreotide could not inhibit the expression of IGF-1 inCNVMs, the other methods were effective.All three medications in group PO and P7 could decrease the positive expression ofVEGF in CNVMs. The most effective method was two methods combined in groupPO or in group P7 with retrobulbar injection. The inhibitive function was relativelyweak in the early stage, increasing gradually. In the late stage, the positive expressionof VEGF in CNVMs kept low level.No significant difference was found on the expression of IGF-1 or VEGF in CNVMsin different groups (P0 us P7) with same medication (retrobulbar injection or twomethods combined) at the same observed point.RT-PCR and Western blotting showed that IGF-1 mRNA and protein or VEGFmRNA and protein were expressed in the sclera-choroid-retina complex. The changesof IGF-1 mRNA or VEGF mRNA expression in diseased control or treated groupswere coincident with immunohistochemical visualization.Laser photocoagulation destructed the ultrastructure of RPE cells, octreotide providedprotection or repairation to RPE cells.Conclusions: Diode laser photocoagulation with high power, small spot size, shortexposure time was used to generate laser spots in the retina, rupture Bruch'smembrane, could induce CNV in C57BL/6.The main distribution of IGF-1 or VEGF was in CNVMs, they play important roles inthe pathogenesis of choroidal neovascularization.Two methods combined or retrobulbar injection of octreotide could effectively inhibitthe positive expression of IGF-1 in CNVMs, decrease the area of CNVMs. Octreotideinhibited paracrine-autocrine secretion of IGF-1 in the endothelial cells or RPE cellsdirectly or through GH-IGF-1 axis.All three medications could effectively inhibit the positive expression of VEGF inCNVMs, decrease the area of CNVMs. Octreotide downregulated VEGF by inhibitingGH-IGF-1 axis, or blocked up the bind of VEGF and its receptor by combining withspecial receptor on the choroidal endothelial cells, or induced apoptosis of endothelialcells. Octreotide could protect or repair RPE cells, indirectly decreased the secrete ofVEGF.