The Role Of The SDF-1/CXCR4 System In The Formation Of Choroidal Neovascularization

Part 1The Development of Experimental Model by 532nm Diode Laser-induced Choroidal Neovascularization in the BN Rats and Its Natural Course StudyPurpose To investigate natural course of choroidal neovascularization (CNV) after 532nm diode laser induction in a Brown Norway rat model. To study the morphological characteristics of experimental CNV in fluorescence angiography, indocyanine green angiography, optical coherence tomography and histopathological examination.Methods Twenty five rats were used in present study. One randomly selected eye was received photocoagulation by 532nm diode laser. Five to eight laser lesions were created around optic disc per eye. Laser parameters used were:a power of 350 mW, a spot size of 75μm and an exposure duration of 0.1 s. In the other eye, no laser photocoagulation was performed. On day 0,7,14,21,28 and 35 after laser photocoagulation, the formation and development of CNV was evaluated by fundus examination and photographs, fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), optical coherence tomography(OCT) and histopathological examination using light microscopy. Fluorescein staining of laser lesions in FFA was graded and central thickness of CNV was measured using light microscopy and analyzed statistically.Results Immediate after laser photocoagulation hyperfluorescence correlated to break of Bruch's membrane could be clearly seen in ICGA. ICGA showed CNV well-developed on day 14 after photocoagulation. On day 14, disciform leakage staining appeared in the FFA, and the score of fluorescein staining maitained stable from day 14 to 28. OCT image showed a disruption of the highly reflective layer corresponded to the RPE, Bruch's membrane and choriocapillaris on the first day after photocoagulation. CNV was seen as a highly reflective area located beneath the subretinal space on day 14 after photocoagulation. The OCT image well corresponded to the histopathological appearance. CNV was ascertained by light microscopy on day 14. No statistically significant difference in central thickness of CNV from day 14 to day 28(P>0.05).Conclusions The present study demonstrated that 532nm diode laser could successfully induce choroidal neovascularization experimental model in the Brown Norway rat. The formation and development of CNV could be well evaluated by FFA, ICGA, OCT and histopathological examination.Part 2The Role of SDF-1/CXCR4 axis and its Associated Cytokines in the Formation of laser-induced CNV in BN ratsPurpose By the laser-induced CNV of BN rats, to measure the expression of mRNA of SDF-1, CXCR4, VEGF-A, CXCR7, and HIF-la from the onset and development of CNV, for exploration its possible molecular mechanism. Methods Established laser-induced CNV of BN rat, respectively, of the samples collected at different time points were done by using immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction (real-time PCR) quantitative analysis of the SDF-1, CXCR4, CXCR7, VEGF and HIF-la within CNV at the level of mRNA expression.Results Immunohistochemistry showed that on the Day 21 after photocoagulation, SDF-1 and VEGF in the RPE layer and the CNV lesions in a large number of expression. There were significantly increased expression of SDF-1, CXCR4, CXCR7, VEGF and HIF within CNV at mRNA level compared with the normal control (P< 0.05). There were significantly increased expression on 7d groups compared with other experimental groups (P<0.05). There was no significantly difference between 14d group to 21d,28d or 35d group (P> 0.05).Conclusion In the model of laser-induced CNV in BN rats, CNV formation may need of SDF-1, CXCR4, CXCR7, VEGF and HIF and other cytokines involved with a variety of cells; the formation of CNV in the "X factor" may be more than one factor including VEGF and SDF-1. There are two axises in the formation of CNV, one is SDF-1/CXCR4 axis, and another is SDF-1/CXCR7 axis. We may call them as SDF-1/CXCR system indicating both. The biological function of SDF-1/CXCR7 axis is not clear yet.Part 3 The Intervention of Bevacizumab and Exogenous SDF-1 in the Formation of laser-induced CNV in BN ratsPurpose CNV was made in a laser-treated BN rats model. By the intravitreal injection of Bevacizumab, to inhibit the formation of CNV, one week after Bevacizumab injection the second intravitreal injection of used the SDF-1, for blocking the VEGF pathway to explore the role of exogenous SDF-1 in the formatin of CNV.Methods Healthy male BN rats were randomly divided into five groups:the group 1, injected with Bevacizumab only, the group 2 was injected with Bevacizumab and SDF-1, the third group was injected with BSS only as control, the fourth group were treated laser photocoagulation as control, and the fifth group as normal control. One eye randomly selected for each rat as the experimental eye. The group 1, and the group 2 of rats were intravitreal Bevacizumab 2μl/eye immediately after photocoagulation. And the third intravitreal injection BSS 2μl/eyes immediately after photocoagulation. After 7 days the group 2 were made intravitreal injection of SDF-1 at lμl/eye. Respectively, on 7d,14d,21d,28d and 35d of the four groups of BN rats were observed by fundus photography, FFA and ICGA. At 14d and 28d after photocoagulation, all rats were randomly selected using light microscopy and real-time PCR detection of mRNA of SDF-1, CXCR4, CXCR7, VEGF and HIF expression.Results In the group of injected Bevacizumab only showed slowdown the development of CNV, while in the group of injected both Bevacizumab and SDF-1 showed not only delay the development of CNV, but also have a tendency to form fibrosis early.