Experimental Research Of Ranibizumab Inhibited Choroidal Neovascularization In Brown Norway Rat

[Research background] Choroidal neovascularization is an important performance form of ocular angiogenesis that related to various eye diseases, such as age-related macular degeneration, idiopathic chorioretinopathy, histoplasmosis diseases, a high degree of myopia-related macular degeneration, eye tumors and eye injury, which AMD became the first place of elderly vision loss in developed country. CNV often involving the macula, has repeatedly caused bleeding, leakage, scarring, and serious damage to central vision. So far, the treatment of CNV-related diseases is still one of the hot field of scientific research. The mechanism of CNV is not clear, the general view that the CNV is more than one generation of cytokines involved in the complex process in which the imbalance between angiogenesis factor and angiogenesis inhibitor factor plays a key role, and the vascular endothelial growth factor is the most important factor for promoting angiogenesis. At present, the anti-vascular endothelial growth factor in the treatment of CNV was emphasized by countries. Ranibizumab is one of the VEGF inhibitor in the treatment of CNV, it is possible for the treatment of VEGF mediated by the neovascular eye diseases. Ranibizumab also known as rhuFabV2, commodity called Lucentis, it is the second generation source of anti-VEGF reorganization mouse monoclonal antibody fragment (recombinant humanized antigen-binding fragment, rhuFab). The author used retinal electrophysiology technology, cell culture in vitro, morphology, pathological and functional classification gene chip research methods, explored the inhibition and mechanism of Ranibizumab in laser-induced choroidal neovascularization of pigmental rats.Part 1 Retinal functional changes after Ranibizumab treatment of krypton laser-induced Brown Norway ratsObjective 1. To investigative wave characteristics and change range of normal Brown Norway rats on electroretinogram and flash visual evoked potential. 2. To explore the effect of vitreous injection with Ranibizumab after krypton laser-induced Brown Norway rats on electroretinogram. Methods 1. To record BN rat electrophysiological waveform with application of F-ERG and F-VEP technology. 2. With fundus krypton laser photocoagulation by 30 points, the establishment of BN-induced choroidal neovascularization in animal models. Modeling after 3 day injection Ranibizumab in the treatment group glass, the proposed use of ISCEV ERG standard five different records, each group observation point in time (3 d, 1w, 2w, 4w) ERG wave. Results 1. With the increase of light intensity, the latency of a, b wave gradually shorten, the amplitude gradually increased of scotopic ERG on BN rats; With the increase of light intensity, the P wave of flash VEP gradually shorten, no significant changes in the amplitude of BN rats. 2. Different points after Laser-induced, there was no dignificant changes in the a, b-latency of Max response ERG, meanwhile a, b amplitude of Max response ERG decreased; Ranibizumab treatment after 4 w, the amplitude of b wave completely back to normal. Conclusion We recorded visual electrophysiology graphics fluctuations and change scope of normal BN rat; Ranibizumab can improve krypton laser-induced BN rats' retinal function.Part 2 Experimental research of Ranibizumab inhibited huvec and RPE cell proliferation and migrationObjective To investigate the effect of Ranibizumab on human vascular endothelial cells and retinal pigment epithelium cells. Methods MTT was used to measure the proliferation of huvec and RPE cells. Transwell was used to measure the migration of huvec and RPE cells. Results 1. Proliferation of huvec and RPE cells was inhibited by Ranibizumab, and with the increase of concentration of Ranibizumab, the cell viability of huvec and RPE cell lines decreased gradually. 2. When the concentration of Ranibizumab was 0.01, 0.1,1 mg/ml , huvec cells migrated to the under well were 112.68±10.03.58.17±6.52.31.67±4.84(P<0.01 vs con), RPE cells migrated to the under well were 162.4±14.5,82.6±11.9,56.3±10.7 respectively. When the concentration of Ranibizumab was 0.1,1mg/ml, migration PRE cells number decreased significantly (P <0.01 vs con ) . Conclusion Ranibizumab can inhibit huvec and RPE cells proliferating and migrating in a clear dose-dependent manner, which may be a way that it inhibits angiogenesis. Part 3 Morphologic research after the treatment of Ranibizumab ofkrypton laser-induced CNV Brown Norway ratsObjective To investigated the morphological changes after the treatment of Ranibizumab of krypton laser-induced CNV BN rats in vivo. Methods Krypton laser-induced CNV BN rats after 2 weeks, application of FFA, HE staining and the Fluorescein isothiocyanate stretched preparation to observe inhibitory effect of Ranibizumab on the CNV. Results Ranibizumab treatment after 2 w, HE staining indicated CNV relative thickness thinning on BN rats; FFA results showed that laser spot reduced leakage score percentile; FITC fluorescent-tagged CNV area reduced. Conclusion Ranibizumab showed the inhibitory effect on the krypton laser-induced CNV of BN rats in vivo.Part 4 The changes of Functional classification Gene expression after Krypton laser-induced CNV and intravitreal injection withRanibizumab in Brown Norway ratsObjective 1. To observe the changes of BN rats' retina-choroidal-sclera complex gene expression of krypton laser-induced CNV at different observation points. 2. To test the changes of BN rats' retina-choroidal-sclera complex gene expression after intravitreal injection with Ranibizumab about 2 weeks. Methods Applied to Category functional gene chips to detect the changes of krypton laser-induced CNV and intravitreal injection with Ranibizumab. Results 1. After 3 day and 1 week of laser photocoagulation, upregulated gene were proinflammatory response factor mostly of CCL, IL, PDGF. After 2 weeks of photocoagulation, promoting angiogenesis factor gene expression increased mainly such as VEGF, TGF, TNF. 2. There are 14 gene upragulated and 11 gene downregualted compared with CNV 2w group after vitreous injection of Ranibizumab. Promoting angiogenesis gene expression upragulated significantly including VEGF, VEGF receptor KDR and TGF. Conclusion 1. In the early stages of Krypton laser-induced CNV (3 d after induction and 1 w), it is ocular inflammation and immune activation play an important role in CNV development. 2. In the early stages CNV (inflammatory phase), mononucleosis (MPC-1-induced) from the cycle into the damaged areas contributed to activation of macrophages, the secretion of a variety of factors to expand inflammatory response. 3. Laser-induced 2 weeks, CNV was formation. The performance of CNV is the activation of EC and RPE cells therefore the release of various promoting angiogenesis genes increased expression, proinflammatory gene reduced or disappeared, involved in the regulation of angiogenesis. The imbalance between vascular growth factor and inhibitive factor is one of the key factors in this stage. 4. After Ranibizumab treatment, effectively blocking the VEGF and its receptor gene expression and thus inhibit the growth of the CNV.