Preliminary Study On Photodynamic Therapy Effect By Hematoporphyrin Monomerthyl Ether On Experimental Choroidal Neovascularization

Background:Choroidal neovascularization(CNV),a common cause of vision loss in patients,often results from exudative AMD,pathologic myopia, angioid streaks,CSC and POHS.Despite extensive basic and clinical researches, The complicated molecular mechanism of CNV is still not completely understood. The currently used therapeutic approaches such as laser photocoagulation, photodynamic therapy(PDT),transpupillary thermotherapy(TTT),submacular surgery are insufficiently effective.BPD-MA-PDT represents a major advance in the treatment of CNV.However it is too expensive to limit its application in China. So it is important to discover a new photosensitizer for the treatment of CNV.Objective:To study the effects of PDT with hematoporphyrin monomethyl ether(HMME),on experimental choroidal neovascularization in the Brown-Norway(BN)rat eye.Methods:1.BN rats were randomly divided inio 7 groups as 3d,7d,14d, 21d,28d,56d,84d groups.Krypton laser photocoagulation was performed on the retina of experimental rats to set up a CNV animal model.The wavelength was 647nm,and the spot size was 50μm.Power delivered ranged from 200 to 260mw, and applied for 0.1 to 0.2 sec.3d,7d,14d,21d,28d,56d,84d after photocoagulation,FFA and HE histology of CNV were analyzed.2.Fluorescein angiography was performed in Wistar and BN rats using a digital fundus camera after 60.57 mg/kg HMME was injected into the femoral vein.The timer was started as soon as the HMME was injected.Peak intensities of HMME fluorescence were determined by visual analysis of the angiograms. Fluorescence microscopy was performed in Wistar rats only.15 mg/kg HMME was injected into the femoral vein.The eyes were enucleated at 5min,20min, 40min,1h,2h and 24h after injection and frozen sections were prepared for viewing under a fluorescence microscope.The intensity of fluorescence was measured with imaging software.Fluorescein angiography and fluorescence microscopy were performed in BN rats with confirmed CNV after HMME was injected.3.PDT was performed in BN rat eyes with experimental CNV induced by laser injury.HMME was administered intravenously 15mg/kg as a bolus. Photodynamic therapy was performed using light at a wavelength of 630 nm,the laser spot size was set at 800-1000μm,with an irradiance of 400,600 and 1000mW/cm~2 and fluence of 36,54,90 J/cm~2.Also HMME was administered intravenously 30mg/kg as a bolus.PDT was performed using light at a wavelength of 630 nm,with an irradiance of 600 mW/cm~2 and fluence of 36,54,90 J/cm~2. Follow-up studies,including FFA,were performed 24 hours or 7 days after PDT. Tissues were examined under light microscope at the end of follow-up. Immunohistochemical visualization of platelet endothelial cell adhesion molecule PECAM-1 was performed in flatmounts,and then the results were analyzed.Results:1.FFA showed retinal edema,disc form fluorescein leakage and more fluorescein leakage 3,7 and 14d after photocoagulation,respectively.After 14d,fluorescein leakage little increased.Histology showed 3d after photocoagulation retinal edema,which was regressed after 7d.CNV were found after 7d.The areas of CNV increased and a great deal CNV was formed after 14d.2.Fluorescein angiography and fluorescence microscopy showed that the maximal fluorescence was deteced at 5min.The extent of fluorexcence was diminished over the course of the study.Fluorescence was somewhat decreased 40min after injection and was not observed after 24h.Fluorescein angiography showed maximal fluorescence in the CNV 20min after injection.The fluorescence was decreased after 40min and not observed after 1h.Fluorescence microscopy showed that the fluorescence intensity of HMME in the CNV increased,reaching its peak at 20min and 40min,then decreased after 1h.By 24h after injection,the fluorescence was subsided in all tissues.3.Rates of the CNV closure varied as a function of the HMME dose and the activating light energy.Angiographic closure of the CNV correlated with damage to the neovascular complex,as seen under light microscope.Damage to areas of choroid and retina treated with HMME-PDT also varied as a function of the HMME and light energy.Endothelial cells labeled by PECAM-1 were present either circular vessel-forming endothelial cells or flat endothelial cells in the center of the scar.CNV lesions were significantly reduced in size(P＜0.01).Conclusion:1.Krypton laser photocoagulation induced CNV in BN rats may be used in studying CNV.14-21d after photocoagulation may be the optimal time to perform PDT.2.Dye pharmacokinetics and localization as detected by fluorescein angiography and fluorescence microscopy can show HMME was quickly concentrated,which can significantly reduce the systematic side effects.The accumulation pattern of HMME in the CNV can demonstrate the highest level of dye accumulation both in CNV and in normal tissue within 20min after dye injection.PDT at this time cannot produce selective occlusion of CNV.The best time presumably would be 20-40min after dye injection.3.PDT with HMME can effectively occlude CNV.Angiographic closure of the CNV lesion in the rat model shows a drug and light dose dependency in the experiment.The collateral damage to the normal tissue also shows a drug and light dose dependency.HMME is a potentially useful photosensitizer for the treatment of CNV.