| Objective1. Observe the expression of mcp-1 and FⅧ-RAg in choroidal neovascularization in laser induced BN rat CNV model by immunohistochemistry during 8 weeks, and investigate the relationship between MCP-1 and CNV development, so propose the opinion that macrophage accumulation play a role in the pathogenesis and maybe a new CNV model can be created according the result in the future. 2.Exam several cytokines' (NF-κB, MCP-1, ICAM-1, VEGF, FⅧl-RAg) protein expression in CNV, meanwhile study the influence of NF-κB on the mRNA transcription of VEGF and ICAM-1, so investigate the relationship between these cytokines and the initiation and development of CNV.3. Study the evolution of CNV during 4 weeks with intravitreal injection of triamcinolone (320μg/8μl), so make a preliminary investigation of the treatment effect of triamcinolone on experimental CNV.4. After the injecton of triamcinolone, observe the protein expression of NF-κB, MCP-1, ICAM-1 and FⅧ-RAg, and exam the transcription of ICAM-1, VEGF on different time points, to research the probable cause of inhibiton of CNV.Method1. Eight groups of 48 BN rats were photocoagulated by krypton laser in one eye to induce CNV. Krypton laser irradiation (647nm) was delivered through a Zeiss slit lamp with-53DS contact lens. The laser spots were placed separately using a setting of 100 μm diameter;0.05second duration, and 360mW intensity. In 48 eyes, 20 laser burns were arranged in the eye, surrounding the optic nerve at the posterior pole. Fluorescein angiography was performed from 1 week to 8 weeks after laser photocoagulation through intraperitoneal injection of0.2-0.3ml of 10% sodium fluorescein. All procedures adhered to the guidelines described in the ARVO statement of the use of animals in ophthalmic and vision research. Six eyes were taken at each time interval. The enucleated eyes were then fixed in 4% paraformaldehyde in 0.1MPBS for 2 hours. Eye cups were embedded in paraffin;Paraffin sections of 5 um thickness were cut and stored at 4°C. All sections were mounted on polylysine coated slides, the protein expression of MCP-1 and F VI -RAg were investigated with immunohistochemistry and were semiquantitatively analyzed with the image pro-plus system.2. Some paraffin sections were deparaffinised, stained with haematoxylin and eosin, and the CNV area was measured using a light microscope;the expression of NF-kB, MCP-1 and ICAM-1 during 8 weeks was examed with immunohistochemistry and observed the gene expression of ICAM-1 and VEGF with in situ hybridization using mRNA probe, and the localization of these cytokines were studied.3. Eight groups of 48 male BN rats received krypton red laser photocoagulation to induce CNV model in one eye, in which 4 groups received intravitreal injection of triamcinolone 8ul (320(xg) immediately after photocoagulation and the other 4 groups received the same volume intravitreally injection of BSS as control groups. Fundus fluorescein angiography examinations occurred on week(s) 1, 2, and 4. 6 eyes were taken at each interval, paraffin sections were made and stained for light microscopy with hematoxylin-eosin, and the area of CNV were measured by 1 reader in a masked fashion.4. In order to study the pathogenesis of triamcinolone inhibiting CNV, the immunostaining of NF-kB, MCP-1, ICAM-1, and F VIE-RAg were analyzed semiquantitatively on paraffin sections, and VEGF and ICAM-1 mRNA signals were examed by in situ hybridization and were semiquantitatively analyzed. ResultI. The immunostaining of FW-RAg shows the persistent increase of the CNVdevelopment during 8 weeks, and the expression of MCP-1 increases in accordance with the CNV development.2. The expression of NF-kB increased during 8 weeks, the expression of MCP-1, ICAM-1, and FVffi-RAg increased as well, and the gene expression of VEGF and ICAM-1 was upregulated.3. The area and the leakage of CNV in triamcinolone treated BN rat eyes obviously reduced compared with the control group at each interval.4. In the treatment group, the expression of NF-kB, MCP-1, ICAM-1, and FVffl -RAg depressed, and the gene expression of VEGF and ICAM-1 wasn't significantly increased. The examination of F VI -RAg and FFA verified the depression of CNV compared with the control group at each interval. Conclusion1. During 8 weeks, the examination of FVI-RAg with immunohistochemistry showed the evolution of CNV, and the expression of MCP-1, which was primarily located in the vascular endothelium, macrophage and RPE cells in the CNV, increased in the corresponding period. As a potent chemokine of monocyte, MCP-1 directively facilitating the neovascularization or indirectively promoting neovascularization by accumlating immunocyte such as macrophage maybe plays an important role in the CNV development.2. During the CNV development, the expression of NF-kB increased gradually from faint situation, and was primarily located in the endothelium and RPE cells of neovascularization, in addition, the expression of MCP-1, ICAM-1, and FVi -RAg increased simultaneously, which hints NF-kB probably is the trigger factor of CNV formation and correlate with expression of MCP-1 and ICAM-1, these upregulated cytokines concomitantly involved in the CNV formation. At the same time, the transcription of VEGF and ICAM-1 increased, so it is speculated that activated NF-kB combines with DNA in nucleous, and then regulates the gene expression of MCP-1, ICAM-1, and VEGF, which sequently triggers and facilitates the CNV formation.3. One eye of every BN rat in treatment group was intravitreal injected triamcinolone acetonide (8ul) imdiately after laser photocoagulation. The area of the CNV in treatment group significantly decreased compared with the control group. In addition, the fundus fluorescein angiography examination showed the neovascularization leakage attenuated. This proved that intravitreal injection of triamcinolone immedialtely after photocoagulation can inhibit CNV development.4. The expression of NF-kB, ICAM-1, VEGF, and MCP-1 is obviously decreased, also the gene transcription of ICAM-1 and VEGF. Triamcinolone acetonide maybe exert CNV inhibition via inhibiting these cytokines5 gene transcription and protein expression. So triamcinolone was verified an efficacious drug for treating CNV.
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