应用血啉单醚光动力疗法治疗角膜新生血管的研究

目的观察应用血啉单醚行光动力疗法(PDT)治疗角膜新生血管的效果。设计前瞻性随机对照实验。研究对象成年有色兔18只。方法制作碱烧伤角膜新生血管模型。血啉单醚5mg/kg静脉注射,不同能量密度(7.6￣152.8J/cm2)的氩绿激光照射角膜新生血管根部,不注射血啉单醚只单纯行同等能量密度的激光照射组作为对照组。主要指标PDT的能量密度,行角膜新生血管荧光素造影观察新生血管封闭情况。结果PDT后3天,角膜新生血管荧光素血管造影显示:应用61.2J/cm2及以上的能量,有67%以上的眼角膜新生血管被完全封闭,33%的眼部分有效。PDT后1周,61.2J/cm2及以上组仍有66.7%(16/24眼)的眼角膜新生血管完全封闭。激光后1个月5/24眼无新生血管出现,其余眼再次出现新生血管。结论采用激光能量密度在(61.2￣152.8)J/cm2照射的PDT治疗能完全或部分封闭兔碱烧伤角膜新生血管模型中的角膜新生血管,但有新生血管再生。(眼科,2006,15:180-183)     

光动力学治疗角膜新生血管

介绍光动力学治疗（PDT）的机制、光敏剂的种类和PDT用于角膜新生血管的研究现状。PDT应用低能量的光作用于光敏剂，产生对靶组织有毒性的光化学反应。光敏剂易于聚积在肿瘤和新生血管处，被增殖性的细胞摄取，这些组织吸收激光能量，导致自身氧化作用和细胞膜、线粒体、溶酶体和核的直接损伤，最终导致靶组织的新生血管和肿瘤组织的细胞凋亡。临床常用的光敏剂多是卟啉和它的衍生物，包括：苯卟啉衍生物单酸、氯化铝酞花青磺酸盐（CASPc）、SnET2、SINc、菌绿素a等。血啉单醚是一种国产的较理想的单体光动力学治疗新药。国外研究应用ATX-S10和SnET 2等作为光敏剂治疗角膜新生血管，疗效显著。     

应用血啉甲醚的光动力学疗法治疗实验性虹膜新生血管

目的观察以国产血啉甲醚(HMME)为光敏剂,应用光动力学疗法(PDT)封闭实验性虹膜新生血管(INV)的疗效和并发症。方法应用氩离子激光封闭猴眼全部视网膜分枝静脉的方法建立INV模型。静脉注射HMME(5 mg/kg),用514 nm的氩绿激光照射,设定能量密度分别为107.46、143.28、275.22 J/cm23组,行虹膜彩像、虹膜荧光造影(IFA)、虹膜吲哚青绿造影(IICGA)及光镜和电镜观察疗效。结果PDT治疗后48 h,治疗区INV基本闭塞;ICGA显示基质层血管有不同程度的闭塞。PDT后3周,治疗区域新生血管仍闭塞,虹膜基质血管重新开放;非治疗区新生血管退行。光镜显示中能量组虹膜基质轻度萎缩,高能量组虹膜可见大量色素增殖。治疗组均未发生眼压升高。结论以HMME为光敏剂的PDT在一定时期内可有效封闭实验性虹膜新生血管,减少新生血管性青光眼的发生,但对虹膜基质有一定损伤。     

光动力治疗兔角膜新生血管的研究

目的：观察光动力学治疗(photodynamic therapy,PDT)缝线法诱导的兔角膜新生血管(cornaeal neovascularization,CNV)的效果和安全性。方法：取新西兰白兔5只10眼,用5．0号的丝线在角膜上缝线诱导角膜新生血管的生长,注射光敏剂Photofrin 48h后行DIOMED630PDT激光照射。裂隙灯显微镜观察角膜NV生长情况,并且计算其面积、生长长度,照射后28d处死实验兔,取其眼球做组织病理学检查。结果：角膜缝线后3d开始有新生血管长入,3wk后长到缝线的尖端。激光照射后2d开始角膜新生血管变得模糊不清,2wk后角膜新生血管近完全消失。组织病理学检查见角膜,视网膜以及虹膜、睫状体、脉络膜等组织正常。结论：光动力治疗角膜新生血管有效、安全。     

应用verteporfin的光动力疗法治疗兔虹膜新生血管的效果观察

目的 建立兔虹膜新生血管模型,观察以verteporfin为光敏剂行光动力疗法(photodynamic therapy, PDT)治疗兔虹膜新生血管(iris neovascularization, NVI)的效果.方法 应用前段缺血法建立新西兰白兔NVI模型,以verteporfin为光敏剂,以689nm二极管激光行PDT治疗,同一眼分治疗区和对照区,每组3眼,用虹膜荧光造影(iris fluorescein angiography, IFA)、光镜、电镜观察治疗后1d(3眼),1周(3眼),1月(3眼)虹膜新生血管改变.正常眼(3眼)为正常对照组.结果 IFA显示PDT治疗后1d及1周治疗区NVI闭塞,治疗后1月治疗区有新生血管重新出现.光镜和电镜显示治疗区1d及1周NVI明显消退,治疗组与正常对照组比较,PDT治疗后NVI周围细胞和组织结构无明显改变.结论 以verteporfin为光敏剂行PDT,可有效治疗兔虹膜新生血管(NVI),但疗效不持久.     

血啉甲醚光动力疗法治疗兔脉络膜黑色素瘤的实验研究

目的探讨国产新型光敏剂血啉甲醚光动力疗法（PDT）治疗兔脉络膜黑色素瘤的效果。方法选择46只新西兰白兔（46只眼）建立脉络膜黑色素瘤模型。每日注射环孢素A以抑制免疫反应,用间接检眼镜和B超观察肿瘤生长,至瘤体为1．5～4．6mm厚度时,将46只兔（46只眼）分成两组,实验组41只眼,均静脉给予血啉甲醚10mg／kg体重,3h后用波长为630nm的氦氖激光进行照射,激光能量60～150J／cm^2。对照组5只眼,其中1只眼为空白对照,2只眼为血啉甲醚对照,2只眼为激光照射对照。PDT治疗后通过间接检眼镜、B超、彩色眼底照相观察瘤体变化4～5周,实验组16只眼分别在激光照射后24h和1周时处死,摘取眼球做病理检查,以观察疗效。结果实验组中,当激光能量≥70J／cm^2时,肿瘤生长受到明显抑制,其厚度明显缩小;在激光能量为60J／cm^2照射下,仅有部分肿瘤生长受到抑制。对照组兔眼肿瘤均在2～3周内迅速增大,甚至充满整个玻璃体腔。结论国产光敏剂血啉甲醚光动力疗法对脉络膜黑色素瘤具有一定治疗作用。     

verteporfin光动力疗法对体外培养的成人视网膜色素上皮细胞色素上皮衍生因子、血管内皮生长因子表达水平的影响

目的 观察经光敏剂verteporfin光动力疗法（PDT）处理后的体外培养的成人视网膜色素上皮（RPE）细胞色素上皮衍生因子（PEDF）、血管内皮生长因子（VEGF）mRNA表达水平的改变。 方法 采用噻唑蓝比色法（MTT）测定PDT前后体外培养的成人RPE细胞活性的变化。应用半定量逆转录聚合酶链式反应(RT-PCR)测定PDT前后RPE细胞PEDF和VEGF mRNA表达水平的变化。 结果 PDT可造成RPE细胞死亡，死亡率与激光能量密度和光敏剂浓度成正相关。PDT后RPE细胞表达PEDF、VEGF mRNA水平下降，下降程度与激光能量密度和光敏剂浓度成正相关。 结论 PDT可下调体外培养的成人RPE细胞PEDF、VEGF mRNA的表达水平，这可能与PDT治疗黄斑下脉络膜新生血管膜（CNVM）后CNVM的消退或再生有关。 (中华眼底病杂志, 2006, 22: 256-260)     

大鼠角膜碱烧伤后碱性成纤维细胞生长因子在角膜中的表达及意义

目的 探讨大鼠角膜碱烧伤后成纤维细胞生长因子（bFGF）在角膜中的表达和意义。方法 采用碱烧伤大鼠角膜建立角膜炎症性新生血管动物模型；免疫印迹法检测大鼠角膜碱烧伤后不同时间段bFGF在角膜中的表达；免疫组织化学方法检测大鼠角膜碱烧伤后bFGF在角膜不同组织层次的表达。结果 免疫印迹法检测显示：去除上皮后的正常大鼠角膜无bFGF表达；碱烧伤后96h，大鼠角膜可检测出bFGF表达，随着时间进展，bFGF蛋白表达量逐渐增加。免疫组化结果：正常大鼠角膜上皮层表达bFGF；碱烧伤后角膜上皮层bFGF着染程度逐渐增强，在碱烧伤后48h以前，基质层浸润的炎症细胞无bFGF着染，96h以后，浸润的炎症细胞和新生血管内皮细胞均呈bFGF阳性。结论 bFGF未参与碱烧伤后角膜新生血管增殖的早期诱导，对角膜炎症性新生血管的增殖仅起到后续支持作用。     

血啉甲醚在兔眼的组织分布

光动力治疗利用光敏剂在新生血管等增生活跃的组织中聚集、滞留的特点．在相应波长的激光照射下产生光化学效应，选择性治疗病变区域，而对周围正常组织损伤很少，逐渐成为治疗脉络膜新生血管的热点。血啉甲醚(hematoporphyrin monomethyl ether．HMME)是国产新型光敏剂．具有光敏化作用强、毒副作用小等优点。本实验观察     

飞秒激光辅助的DLK治疗角膜碱烧伤后角膜组织病理学及超微结构观察

背景眼表结构与功能的正常与稳定是维持角膜透明及视功能的重要保证,在眼化学烧伤传统治疗的基础上对早期眼化学烧伤提出新的治疗理念、掌握合适的手术时机和娴熟的手术技巧可减少碱性物质对角膜及眼内组织的进一步损害。目的观察飞秒激光辅助的深板层角膜移植术（DLK）治疗角膜急性碱烧伤的组织病理学及超微结构变化,探讨角膜碱烧伤早期DLK治疗的可行性及疗效。方法采用12只成年健康新西兰白兔,将浸有1mol／LNaOH溶液30斗l的直径6mm无菌滤纸片置于中央角膜30s,建立角膜碱烧伤模型。模型兔用随机数字表法分为2个组,飞秒激光辅助的DLK治疗组（治疗组）于角膜碱烧伤后24h采用飞秒激光辅助的DLK将家兔角膜植片移植到新西兰白兔模型眼,模型对照组不施加任何干预和治疗。分别于术后第1、2、4周各组各处死2只实验兔,将获取的角膜组织进行常规固定和包埋,然后制作切片,行苏木精一伊红染色,分别在光学显微镜和透射电子显微镜下观察角膜的组织病理学变化和超微结构变化。结果造模后可见烧伤区域角膜立刻变为瓷白色,结膜充血。术后4周,裂隙灯显微镜下可见治疗组兔眼角膜植片透明,而模型对照组兔眼角膜水肿,可见角巩膜缘结膜充血和少量新生血管。角膜组织病理学检查可见模型对照组角膜基质层水肿,胶原纤维排列疏松,纤维间可见大量空泡和少量新生血管,并有大量炎性细胞浸润,治疗组仅见角膜植片轻度水肿、透明,组织间未见炎性细胞浸润。透射电子显微镜观察可见,术后第1周治疗组兔角膜上皮细胞层中细胞间桥粒连接较多,细胞核完整;术后第4周角膜中央透明,基质中胶原纤维排列整齐,可见成纤维细胞;而模型对照组术后第4周可见角膜上皮表层扁平细胞的存在,部分上皮细胞脱落,形成     

Accumulation of photosensitizer ATX-S10 (Na) in experimental corneal neovascularization

PURPOSE: To determine the most appropriate time for laser irradiation to produce selective occlusion of new corneal vessels by photodynamic therapy (PDT) with a new photosensitizer, ATX-S10(Na). METHODS: The time course of the plasma levels of ATX-S10(Na) and the degree of dye accumulation in the corneal neovascularization after intravenous administration was determined in rabbit eyes. Plasma concentration of ATX-S10(Na) was analyzed by a spectrophotometer. The amount of ATX-S10(Na) in the new corneal vessels was measured by nitrogen-pulsed laser spectrofluorometry. Frozen sections of neovascularized cornea and iris were observed by fluorescence microscopy. RESULTS: Plasma ATX-S10(Na) concentration was highest 5 minutes after dye injection and rapidly decreased and reached almost zero at 24 hours, indicating its prompt excretion from the body. The amount of ATX-S10(Na) in the new corneal vessels as measured by nitrogen-pulsed laser spectrofluorometry increased and reached maximal level at 2 to 4 hours. Under fluorescence microscopy, the dye was more abundantly localized in the wall of new corneal vessels than in the normal tissue at 2 to 4 hours. CONCLUSION: These results indicate that laser irradiation between 2 and 4 hours after dye injection is appropriate for selective PDT with ATX-S10(Na) for the occlusion of new corneal vessels.     

Treatment parameters for selective occlusion of experimental corneal neovascularization by photodynamic therapy using a water soluble photosensitizer, ATX-S10(Na)

Time dependent change of an accumulation of an amphiphilic photosensitizer, ATX-S10(Na) on rabbit corneal neovascularization (CoNV) was evaluated by angiography using ATX-S10(Na) as a fluorescent dye on three rabbits. The angiography showed that the dye accumulated on CoNV 3-5 hr after dye injection when the dye in the iris was minimum. The results suggested 3-5 hr after might be the optimal time to start photodynamic therapy (PDT) to occlude CoNV selectively without damage to the surrounding normal tissue such as the iris. Then the optimal treatment parameters in PDT using ATX-S10(Na) for selective occlusion of the CoNV were investigated on rabbit eyes. PDT was performed with two different time intervals between dye injection and laser irradiation of a diode laser (670 nm), different laser doses and three different dye doses on 21 animals. PDT performed immediately after dye injection selectively occluded CoNV with laser irradiations from 30.6 to 38.2 J cm(-2)and a 2 mg kg(-1)dose of ATX-S10(Na), as well as with 15.3 J cm(-2)and a 6 mg kg(-1)dose. PDT performed 4 hr after dye injection with 107.0-152.8 J cm(-2)and a 6 mg kg(-1)dose, as well as with 38.2-53.5 J cm(-2)and a 12 mg kg(-1)dose was also effective. Although PDT performed either immediately or 4 hr after ATX-S10(Na) injection selectively occluded CoNV, the width of the optimal range of radiant exposures seemed wider in PDT performed 4 hr after dye injection. It is supposed that this result is associated with the difference of dye accumulation between in CoNV and in normal tissue as shown by the present angiographical findings.     

维替泊芬介导的光动力疗法治疗兔眼角膜新生血管

目的:旨在探讨和评估光敏剂维替泊芬(vertporfin)介导的光动力疗法(PDT)对角膜新生血管(CoNV)治疗的效果。方法:新西兰白兔随机分为2组:两组采用角膜基质层缝线的方法诱导CoNV形成。Ⅰ组行vertporfin-PDT治疗CoNV,verteporfin以1.5mg/kg静注。Ⅱ组不治疗,为阳性对照。治疗后裂隙灯显微镜观察CoNV变化并记录面积,取处理组及对照组的角膜、虹膜睫状体组织,行组织病理学检查,免疫组织化学(SABC法)检测血管内皮生长因子(VEGF)在角膜组织中的表达。结果:治疗后3d;1,2wk,Ⅰ组CoNV面积均明显小于Ⅱ组(P<0.01)。组织病理学检查显示,Ⅰ组CoNV管壁破坏,形成血栓。VEGF的表达Ⅰ组明显低于Ⅱ组(P<0.01)。结论:Vertporfin-PDT对兔模型眼CoNV有明显的抑制作用,不损伤邻近的正常血管及组织。     

Dose-related structural effects of photodynamic therapy on choroidal and retinal structures of human eyes

PURPOSE: To determine the effects of photodynamic therapy (PDT) on choroidal and retinal structures of human eyes. METHODS: One eye from each of three patients with large malignant melanomas of the uvea destined for enucleation received PDT using verteporfin according to the approved treatment recommendations for patients with age-related macular degeneration. Two laser spots and two light doses (50 J/cm(2) and 100 J/cm(2)) were applied in unaffected chorioretinal areas. The effects of PDT were assessed by fluorescein and indocyanine-green angiography. The eyes were enucleated 1 week later, fixed in buffered paraformaldehyde/glutaraldehyde solution, bisected along the laser spots, and processed for light and electron microscopy. RESULTS: In agreement with the clinical angiographic findings of hypofluorescence, a rather selective occlusion of the choriocapillary layer was observed in the 50-J/cm(2) PDT areas, whereas the 100-J/cm(2) PDT areas additionally revealed closure of deeper choroidal vessels and focal alterations of the retinal pigment epithelium. The overlying neurosensory retina, including photoreceptors and retinal capillaries, was well preserved in all PDT areas. Electron microscopy showed that alterations of the choriocapillary endothelium comprised swelling, shrinkage and fragmentation of endothelial cells, detachment from their basement membrane up to complete degeneration of the endothelial lining, leading to platelet aggregation, degranulation, and thrombus formation. Complete occlusion of capillary lumina by fibrin, thrombocytes, and cellular debris was observed. Remaining intact endothelial cells appeared to be reorganized into novel smaller vascular channels within occluded lumina. CONCLUSIONS: PDT with verteporfin at a dosage used clinically induces selective occlusion of the physiological choriocapillaris without affecting deeper choroidal, retinal, and optic nerve vessels or the overlying retinal pigment epithelium and neurosensory retina. The main mechanism of action appears to be vascular thrombosis induced by cytotoxic damage of endothelial cells and platelet activation. An increase in light dose enhances the occlusive effect with thrombosis within deeper choroidal layers and damage to the retinal pigment epithelium. However, photoreceptors remained intact at all light doses used.     

The effect of intraocular lidocaine in white rabbit eyes

PURPOSE: Recently, intraocular lidocaine anesthesia has been used in cataract surgery. We studied the toxicity of intraocular unpreserved lidocaine for corneal endothelial cell and retina using Japanese white rabbits. METHOD: They were divided into two groups. One group was injected intracamerally and the other group was injected intravitreally with 0.2 ml of unpreserved lidocaine of 0%, 0.02%, 0.2%, or 2% concentration. The number of corneal endothelial cells was measured 1 week after the injection. The rabbits were killed after measurements, and their corneas were studied histologically. The retina was examined by electroretinogram from before the injection through 1 week after the injection. RESULTS: There was no significant change in number of corneal endothelial cells after injection of 0.2% lidocaine. However, histological abnormality was seen in corneal endothelial cells after 2% lidocaine injection. There was also significant change in electroretinogram with 2% lidocaine injection. No histological abnormality was seen in the retina 1 week after the injection. CONCLUSION: The rabbit cornea and retina manifested no serious changes after the injection of lidocaine at less than 0.2% concentration functionally and histologically.     

The Effect of Intraocular Lidocaine in White Rabbit Eyes

Purpose: Recently, intraocular lidocaine anesthesia has been used in cataract surgery. We studied the toxicity of intraocular unpreserved lidocaine for corneal endothelial cell and retina using Japanese white rabbits.Methods: The rabbits were divided into two groups. One group was injected intracamerally and the other was injected intravitreally with 0.2 ml of unpreserved lidocaine of 0%, 0.02%, 0.2%, or 2% concentration. The number of corneal endothelial cells was measured 1 week after the injection. After measurements, the rabbit corneas were studied histologically. The retina was examined by electroretinogram prior to initial injection through 1 week after the injection.Results: There was no significant change in number of corneal endothelial cells after injection of 0.2% lidocaine. However, histological abnormality was seen in corneal endothelial cells after 2% lidocaine injection. There was also significant change in electroretinogram with 2% lidocaine injection. No histological abnormality was seen in the retina 1 week after the injection.Conclusion: The rabbit cornea and retina manifested no serious changes after the injection of lidocaine at less than 0.2% concentration functionally and histologically.     

Ultrastructural pathology of corneal neovascu-larization after photodynamic therapy in rabbits

AIM: To investigate the ultrastructural pathogenesis of photodynamic therapy (PDT) for the experimental corneal neovascularization (CNV) by Hematoporphyrin Derivate (HPD) as photosensitizer and Argon laser as light source. METHODS: Experimental CNV models were induced in 7 white rabbits using alkali burn. Six weeks after models establishment, animals with CNV were injected with HPD intravenously, and 48 hours after the injection, 7 eyes were irradiated with argon laser (power 800mw, wavelength 514.5nm, spot diameter 200μm, exposure time 2ms). The irradiated CNV was observed by light microscopy and scanning electron microscopy. RESULTS: Histopathological study indicated that there was a striking decrease in the number of the CNV, vascular endothelium became degeneration and necrosis, some vessels were atrophy and attenuated, and vessels cavity were blocked by some thrombosis. No obvious abnormal histopathological findings were noted in surrounding tissues. CONCLUSION: The high precise action on CNV and minimal damage to surrounding tissues with PDT by HPD as photosensitizer suggested that PDT might be an effective and safe modality in the treatment of CNV.     

Histological findings of surgically excised choroidal neovascular membranes after photodynamic therapy

AIM: To investigate effects of photodynamic therapy (PDT) on human choroidal neovascularisation (CNV). METHODS: Two patients with recurrences after PDT with verteporfin underwent surgical extraction of the CNV. Immediately after surgical excision the subfoveal neovascular membranes were divided for light microscopic and for electron microscopic processing. For light microscopy tissues were embedded in paraffin. Sections were stained with haematoxylin and eosin, and the periodic acid Schiff (PAS) reaction was performed to determine histological diagnosis and to ensure tissue quality. For electron microscopy the specimens were fixed in glutaraldehyde and embedded in epoxy resin. Semithin sections were stained with uranyl acetate and lead citrate and examined with a transmission electron microscope. RESULTS: Light microscopy showed thick fibrovascular membranes in both cases. On the outer surface remnants of retinal pigment epithelial cells resting on thickened inner aspect of Bruch's membrane were found. On the retinal side some outer segments were found. The membrane showed areas with irregularly shaped vessels. Electron photomicrographs showed occluded vessels within the CNV containing thrombotic masses and/or ultrastructural damage of the neovascular endothelium. Most of the vessels presented regressive changes with vacuolisation and fragmentation of the neovascular endothelium accompanied by disintegration of the endothelial cell layer. Extravasation of red blood cells was observed. Occasionally, vessels with normal endothelium containing intact red blood cells were observed. Some vessels contained immature endothelial cells. At some locations the retinal pigment epithelium cells (RPE) were metaplastic showing highly vacuolised cytoplasm. CONCLUSIONS: These findings suggest that the evidence of fluorescein leakage from the CNV and enlargement of the neovascular complex following PDT could be related to new vessel growth and recanalisation of occluded vessels. Additionally, RPE disturbances were observed in the specimens. This finding may be related to the original pathology or could indicate that PDT treatment may result in RPE atrophy.     

Clinicopathologic studies of age-related macular degeneration with classic subfoveal choroidal neovascularization treated with photodynamic therapy

BACKGROUND: Photodynamic therapy (PDT) is a relatively new modality that is currently under clinical and experimental evaluation for treatment of subfoveal choroidal neovascularization (CNV). The authors report the case of an 82-year-old woman who underwent verteporfin-mediated PDT for classic subfoveal CNV. Fluorescein angiography performed 2 weeks after treatment disclosed reduction of the initial area of neovascularization and leakage by approximately 60%. Three weeks after PDT, however, the area of leakage was almost the same size as that before treatment. The patient underwent submacular membranectomy almost 4 weeks after treatment. The authors describe the ultrastructural vascular changes after PDT and a clinicopathologic study of classic CNV. METHODS: The submacular membrane was studied by light and electron microscopy and immunohistochemical techniques. RESULTS: Ultrastructural examination of the peripheral vessels showed evidence of endothelial cell degeneration with platelet aggregation and thrombus formation. Occasional occluded vessels were surrounded by macrophages, a phenomenon previously reported to describe the process of resorption of such blood vessels. The vessels in the center of the membrane were unremarkable. CONCLUSION: Photodynamic therapy causes endothelial cell damage, thrombus formation, and vascular occlusion of classic CNV in age-related macular degeneration.