应用半导体激光诱导鼠脉络膜新生血管模型

目的 评价半导体激光诱导C57BL-6J小鼠脉络膜新生血管（choroidal neovascularization,CNV)模型的可行性。方法 对16只小鼠（31只眼）分别于激光照射后2h,3d,1,2及4周行荧光素眼底血管造影(fundus fluorescence angiography,FFA),光镜及电镜检查,以观察激光诱导CNV的效果。结果 激光照射后2h及3d无CNV生成,FFA显示无荧光渗漏,激光照射后1周出现CNV,FFA示1周时荧光渗漏48个点（53.3%),4周时荧光渗漏33个点（36.7%),且CNV出现不一军伴有荧光渗漏,病理切片证实,光凝后1,2及4周,小鼠CNV发生率分别为63.6%,63.6%及66.7%。结论 通过半导体激光光凝诱导C57BL-6J小鼠CNV模型是可行,有效的。     

Matrigel诱导实验性脉络膜新生血管的实验研究

目的：建立兔眼脉络膜新生血管(CNV)的模型,为治疗CNV相关疾病的药效学实验奠定基础。

方法：采用青紫蓝兔视网膜下注射不同浓度Matrigel的方法建立实验性CNV模型,通过眼底彩照、FFA、脉络膜铺片、组织病理切片分别进行评价分析,确定Matrigel最佳浓度及操作方法。

结果：从FFA来看,Matrigel 40μL和20μL组从7d起出现荧光渗漏,14d和28d逐渐增强; 10μL组14d和28d出现荧光渗漏,5μL组一直未见明显荧光素渗漏。从眼底情况来看,7d时大部分注射部位的视网膜已贴附回去,仅个别眼出现局限性视网膜脱离,Matrigel 40μL组个别眼视网膜增殖明显,造成牵拉性视网膜脱离。因白内障或玻璃体混浊无法进行眼底彩照和造影多出现在Matrigel 40μL组。从组织病理学观察,小血管和/或幼稚纤维组织增生多见于Matrigel 40μL和20μL组。但Matrigel 40μL组视网膜损伤明显。

结论：Matrigel视网膜下注射可成功诱导CNV形成,20μL为最佳剂量。     

实验性脉络膜新生血管的定量评价

目的 评价氪激光诱导下大鼠脉络膜新生血管(choroidal neovascularization,CNV)面积计算的可行性.方法 采用氪激光击破大鼠右眼 Bruch 膜的方法诱导24只大鼠产生实验性 CNV,另一眼作正常对照眼.眼底彩照及眼底血管荧光造影检查(fundus fluorescein angiography,FFA)分别于光凝后1周、2周、3周、4周时观察大鼠眼底CNV渗漏情况,脉络膜铺片技术测量CNV面积.结果 氪激光大鼠CNV模型成模率3周、4周时稳定(75.00%、78.75%).从FFA、脉络膜血管铺片技术可见大鼠CNV面积随造模后时间延长呈明显递增生长(P＜0.05),3周、4周时差异不明显,病理切片亦可见CNV中新生血管数量的增多.结论 FFA检查、脉络膜血管铺片技术、病理切片均可作为评价CNV生长的指标,其中脉络膜血管铺片技术是CNV定量评价的可靠方法.     

激光诱导色素兔脉络膜新生血管模型

目的评价半导体泵浦的倍频Nd：YAG532nm绿激光诱导色素兔脉络膜新生血管（choroidal neovascularization．CNV）模型的可行性。方法将24只色素兔按光凝后1周、2周、3周、4周分为4组,每组6只。用高强度倍频Nd：YAG532llm绿激光光凝色素兔视网膜,分别于激光照射后第1、第2、第3及第4周行荧光素眼底血管造影（fundus fluorescein angiography,FFA）,FFA检查后处死动物,取光凝区眼球壁制作标本．进行光镜和透射电镜观察及CD34免疫组织化学染色,以观察激光诱导CNV的效果。结果光凝后第1周．FFA检查见脉络膜期及动脉早期10．0％的光斑有点状荧光素渗漏;光凝后第2、第3、第4周,依次有37．5％、51．7％、45．0％的光斑出现脉络膜新生血管形态。病理切片及CD34免疫组化染色证实,光凝后第1、第2、第3、第4周,兔CNV发生率分别为33．3％、57．1％、66．7％及61．5％。结论通过半导体泵浦的倍频Nd：YAG532nm绿激光诱导色素兔CNV模型是可行的。     

小鼠的脉络膜新生血管模型

目的建立激光诱导的小鼠的脉络膜新生血管模型,观察其自然演变过程.方法通过氩激光光凝诱导C57BL/6J小鼠产生脉络膜新生血管,光凝后1 wk、2wk、4wk、2 mo、3 mo、4mo、5mo、6 mo行荧光素眼底血管造影检查,并且在光凝后1 wk、2wk、4wk、2mo、3 mo、6 mo处死小鼠(n=2),摘除眼球,行组织学检查.结果光凝后1 wk、2 wk、4 wk、2 mo、3 mo、4mo、5mo、6 mo荧光素眼底血管造影显示有荧光渗漏的光凝斑的百分率分别为59.0%(46/78)、62.1%(41/66)、44.4%(24/54)、42.9%(18/42)、46.7%(14/30)、44.5%(8/18)、38.9%(7/18)、22.2%(4/18),组织学检查显示光凝斑内有新生血管形成,随着时间的延长,视网膜色素上皮细胞呈不同程度增生,可完全包绕脉络膜新生血管(choroidal neovascularization,CNV).结论氩激光光凝可成功诱导小鼠的CNV模型,CNV在光凝后2wk达到高峰.     

不同能量半导体激光对大鼠脉络膜新生血管形成的影响

目的 观察不同能量的半导体激光对诱导棕色挪威(BN)大鼠脉络膜新生血管(CNV)形成的影响.方法 雄性BN大鼠42只随机分组,将其中的36只根据激射激光的功率不同平均分为3个实验组,其余6只作为正常对照组.采用810nm半导体激光光凝大鼠双眼视网膜,光斑直径75μm,曝光时间0.1s,激光功率分别为120、140和160mW,于光凝后1、7、14、21、28、56d进行荧光素眼底血管造影(FFA)和吲哚菁绿血管造影(ICGA)检查,随后于每个时间点每组各处死2只大鼠,摘除眼球在光学显微镜下检查CNV形成情况.结果 不同能量半导体激光光凝BN大鼠视网膜后7dCNV开始形成,21d达高峰,此时根据FFA和ICGA计算的3组CNV的成模率分别为51.3%、91.8%、88.3%及51.3%、92.7%、93.7%,56d时CNV有所减少.光凝后7d可见光凝斑周围炎性细胞增多,CNV形成;14d光凝斑增厚,色素细胞增生、移行;21d时140mW及160mW组多数光凝斑增厚,可见明显CNV;随后炎性细胞减少,胶原纤维增多,56d时仍可见CNV结构.结论 功率分别为120、140、160mW的810nm半导体激光均能成功诱导BN大鼠形成CNV,140mW组成模率高且受干扰因素少,是建立BN大鼠CNV模型比较理想的参数.     

氪激光诱导的大鼠脉络膜新生血管模型研究

目的 探讨氪激光创建棕色挪威(brown norway,BN)大鼠脉络膜新生血管(choroidal neovascularization,CNV)模型的可行性,为明确CNV的发生机制及防治研究奠定基础。方法 雄性BN大鼠33只(66只眼),取每只鼠一只眼作为实验眼,另一只眼为对照眼。用氪激光(波长为647nm)对大鼠实验眼视网膜进行光凝,激光功率、光凝斑直径和曝光时间分别为360mW、50μm及0．05s,每只眼10个光凝斑点。于光凝后3、7、14、21、28及56d行荧光素眼底血管造影(fundus fluorescein angiography,FFA)和吲哚青绿血管造影(indocyanine green angiography,ICGA)检查后处死动物,摘除眼球制作标本,进行组织病理学及电镜观察。结果 光凝后7d出现CNV,21d达高峰,造模成功率为76．0％,FFA检查显示典型圆盘状荧光渗漏,ICGA检查可见CNV充盈,光镜和电镜下均可见视网膜下CNV形成。CNV厚度自光凝后7—21d逐渐增加,之后CNV厚度变化不显著。结论 氪激光损伤可以创建BN大鼠脉络膜新生血管模型,成模时间短,持续时间长,成模率高。     

氪激光诱导大鼠脉络膜新生血管基因芯片研究

目的运用基因芯片技术研究氪激光诱导大鼠脉络膜新生血管的差异表达基因．为脉络膜新生血管的治疗提供实验依据。方法氪激光光凝视网膜后7d，分剐提取实验组和对照组脉络膜组织的总RNA，将纯化后的mRNA进行反转录，制备杂交探针，应用含有5705条Oligo DNA的表达谱芯片对实验组和对照组脉络膜组织进行差异表达基因检测。结果氪激光诱导脉络膜新生血管组织的差异表达基因共有427条，其中上调基因为189条，下调基因为238条。上调最高的基因是MMP12基因。在差异表达基因中有代谢、细胞粘附、细胞运动、发育、运输、信号传导、分化等基因。结论氪激光诱导脉络膜新生血管的发生发展涉及多基因改变。[眼科新进展2005；25（5）：408—410]     

激光诱导有色大鼠脉络膜新生血管的形态学观察

目的观察激光诱导有色大鼠脉络膜新生血管(choroidal neovascularization,CNV)的形态学变化,为CNV机制及治疗的研究提供科学的平台和有效客观的评价方法。方法雄性BN大鼠52只(104眼),每只鼠一只眼作为实验眼,另一只眼为对照眼。532nm倍频激光光凝,激光功率、光斑直径和曝光时间分别为140mW、75μm和100ms,每眼光凝8~10点。光凝后1d、3d、7d、14d、21d、28d、56d及110d行荧光素眼底血管造影和吲哚青绿血管造影检查后处死动物,摘除眼球制作标本进行组织病理学观察,并于光凝后3d、7d和56d行透射电镜观察。结果光凝后3d光斑处有内皮细胞的增生,CNV于7d开始形成,14d达高峰,造模成功率为75%.荧光素眼底血管造影显示,造影早期可清晰判断CNV形态,晚期典型圆盘状荧光素渗漏。吲哚青绿血管造影检查可见CNV充盈。光镜和电镜下均可见CNV形成。CNV厚度自光凝后7~14d逐渐增加,之后变化不显著。Ⅷ因子标记的CNV血管密度7~14d逐渐增加,之后变化不显著,56d后逐渐减低。结论激光诱导有色大鼠CNV模型,成模时间短,成模率高,观察时间长,是一种较为理想的实验模型。     

激光诱导小鼠脉络膜新生血管模型中CD105的表达

目的通过激光诱导C57BL／6J小鼠CNV动物模型，观察CD105在新生血管组织中的表达。方法二极管激光诱导生成小鼠脉络膜新生血管，激光后1周和4周以免疫组化观察CD105的表达。结果激光后1周，视网膜下新生血管形成，CD105表达于CNV组织。激光后4周，当血管形成趋于静止时，CD105表达明显减弱。结论研究提示CD105在脉络膜新生血管形成过程中可能起重要作用。     

Study of krypton laser-induced choroidal neovascularization in a Guinea pig model of high anisometropia

 PURPOSE:To investigate the association between high anisometropia and the area of choroidal neovascularization (CNV) induced by krypton laser in guinea pigs and better understand the pathogenesis and prevention of myopic CNV. METHODS:Nine 3-week old male guinea pigs with anisometropia >6.00D were randomly assigned to three groups according to examination date after laser photocoagulation (7d, 14d and 28d). All animals underwent refraction. The eye with higher myopia was used as the experimental eye, and the other as the control eye. All eyes received repeated multi-wavelength krypton laser photocoagulation treatments (wavelength: 532nm; laser power: 400mW; spot diameter: 50μm; exposure time: 0.1s). Fundus photography and indocyanine green angiography (ICGA) were performed. Afterwards, the animals were sacrificed immediately, and the eyes were enucleated and processed for histopathologic examination and flat mounts. RESULTS:CNV appeared at 7d after laser treatment. The area of CNV peaked at 14d, and decrease in area and the presence of scarring was noted at 28 d. CNV was present in 66.7% of eyes by ICGA at 14 d. CNV could be observed under light microscopy at all three time points. At 14d, flat mount showed the neovascular plexus around the lesion. Semi-quantitative analysis revealed that the area of CNV in treated eyes was greater than that of control eyes. CONCLUSION:Since the mechanism of CNV in this study resembles that of CNV in pathological myopia, this model can be used to investigate the etiology, pathogenesis and treatment of CNV in pathological myopia.     

Quantitative image analysis of laser-induced choroidal neovascularization in rat

Rodent models of laser-induced choroidal neovascularization (CNV) are now extensively used to identify angiogenic proteins, determine the role of specific genes with knockout mice, and evaluate the efficacy and safety of anti-angiogenic therapies. CNV is typically evaluated by fluorescein angiography or vascular endothelial cell labeling in histologic sections. The current study examined an alternative method using high molecular weight FITC-dextran (MW 2 x 10(6)) for high resolution angiography in RPE-choroid-sclera flat mounts. At 24 hr after lasering, the lesions appeared as a circular weakly fluorescent area of approximately equal diameter to the laser spot. No FITC-dextran labeled blood vessels were visible in the lesion at day 1. Three days after lasering, 47% of the lesions showed FITC-dextran labeling indicative of CNV. The incidence (71%) and extent of CNV increased by day 6, and by day 10 all lesions were vascularized, and the maximal area was attained. No significant change followed day 10, and the neovascular area remained constant through day 31. The highest rate of blood vessel growth (between 3 and 10 days after laser) correlates with the peak expression of VEGF, bFGF, and their receptors shown in previous studies. Morphologic analysis of flat mounts and histologic sections showed that the neovascular plexus in most lesions originates from deeper choroidal vessels in the center of the lesion, grows towards the neural retina, then branches circumferentially to anastamose with uninjured choriocapillaris. The microvessels in these lesions are broad and flat, similar to normal choriocapillaris. In a separate study, rats were treated daily with the angiostatic corticosteroid dexamethasone (20-500 microg kg(-1)day(-1)), and CNV was examined at day 10 in FITC-dextran labeled flat mounts and histologic sections. Dexamethasone dose-dependently inhibited CNV, and its highest dose inhibited approximately 95% of CNV labeled by FITC-dextran and resulted in lesions with no detectable Factor VIII immunostaining. High resolution angiography with FITC-dextran is reproducible and quantifiable, and it may accelerate the discovery of therapeutic agents that modulate choroidal neovascularization.     

激光诱导小鼠脉络膜新生血管中补体C3的作用

目的探讨激光诱导脉络膜新生血管(choroidal neovasculature,CNV)形成机制中补体的作用。方法用氪红激光对小鼠行实验性视网膜激光光凝,建立CNV动物模型,共聚焦显微镜下观察CNV发生情况,检测补体溶血活性(CH50),用免疫组织化学方法检测补体C3,用RT-PCR分析C3a受体mRNA的表达。结果激光后第7天,对照组C57BL/6小鼠见CNV形成,而眼镜蛇毒因子(cobra venom factor,CVF)预处理组及C3-/-小鼠组未见CNV形成。CVF预处理组小鼠补体的CH50于激光后第1天、第3天、第5天、第7天分别为2%、3%、3%、2%,同时间点对照组均为100%,差异均有显著统计学意义(均为P<0.001)。激光后第1天,对照组RPE-脉络膜-巩膜复合体见C3强阳性染色,CVF预处理组及C3-/-小鼠组未见C3阳性染色。激光后对照组C57BL/6小鼠较激光前,C3a受体mRNA表达增强,激光后第5天,C3a受体mRNA水平达到高峰,并在激光后第7天维持较高水平,而CVF预处理组小鼠及缺乏补体C3的C3-/-小鼠组激光前后未见明显改变。结论氪激光诱导CNV模型,补体C3参与CNV形成。补体系统的存在和激活是激光诱导CNV形成的必要条件。     

经瞳孔温热疗法治疗隐匿性脉络膜新生血管

目的:对经瞳孔温热疗法(transpupillary thermotherapy,TTT)治疗隐匿性脉络膜新生血管(occult choroidal neovas-cularization,OCNV)的效果进行评价。方法:眼底荧光素血管造影(fundus fluorescence angiog-raphy,FFA)及吲哚青绿血管造影(indocyanine green angiography,ICGA)检查后确诊为单眼或双眼OCNV患者35例43眼,使用Iris810nm半导体激光进行TTT治疗,选择1.2,2.0及3.0mm直径的光斑,以光斑大小能够完全覆盖FFA及ICGA所显示的病变范围为宜,长脉冲模式,能量80~300mW,照射时间60s。结果:TTT治疗后,18/43眼(42%)视力提高,23/43眼(53%)视力无变化,2/43眼(5%)视力下降;20/43眼(47%)CNV渗漏完全停止,18/43眼(42%)渗漏明显减少,4/43眼(9%)渗漏无改变,1/43眼(2%)渗漏扩大。结论:TTT治疗OCNV效果较好,其适宜的参数及治疗机制还需进一步探讨。     

Inhibition of TNF-alpha reduces laser-induced choroidal neovascularization

To investigate the role of the TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in a mouse model. Four separate laser burns were applied to induce ruptures of Bruch's membrane and subsequent choroidal neovascularization in C57BL/6J mice. TNF-alpha protein expression was semiquantitatively assessed by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. To investigate the effect of TNF-alpha inhibition on CNV formation, animals were treated for 7 days via intraperitonealy implanted osmotic pumps either 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept), a chimeric monoclonal antibody (infliximab), or a purified rat anti-mouse/rat TNF monoclonal antibody (TNF-mAb), respectively. Fluorescein angiography, flat-mount preparations, and histopathology were performed at day 7, 10, or 14 after laser treatment. Western blotting demonstrated that TNF-alpha expression was 4.57-fold higher in the choroid and RPE one week after laser injury compared to control mice without laser. When evaluated one and two weeks after laser injury, etanercept and infliximab given from the 3rd day before laser-damage significantly reduced CNV size and pathological fluorescein leakage compared to the control group after laser treatment only. The inhibitory effect of the monoclonal TNF-alpha antibody on CNV formation was evident two weeks after photocoagulation but not after one week. Only etanercept administered 3 days after laser injury still reduced significantly the development of CNV lesions. Histopathology confirmed that CNV lesions in treated mice were smaller in size compared to the control animals without TNF inhibitor treatment. In conclusion, anti-TNF-alpha treatment with different inhibitors reduces both the size and the leakage of laser-induced CNV. These results suggest the involvement of TNF-alpha in the development of laser-induced CNV and its potential use as a therapeutic agent in the age-related macular degeneration.     

重组人血管内皮抑素对脉络膜新生血管的抑制作用

目的 探讨重组人血管内皮抑素对半导体激光诱导的Brown Norway大鼠脉络膜新生血管(choroidal neovascuIariza-tion,CNV)的抑制作用.方法 取雄性大鼠30只(60眼),大鼠双眼眼底采用半导体激光光凝建立CNV模型,随机分为3组:实验组、生理盐水组及空白对照组,每组10只(20眼).实验组玻璃体内注射重组人血管内皮抑素20 μL(5g·L-1),每2d注射1次,共注射5次;生理盐水组玻璃体内注射生理盐水20μL,每2d注射1次,共注射5次;空白对照组光凝后不做处理,观察期为14d.观察术后13d各组大鼠荧光素渗漏情况,术后14d光镜下观察组织细胞学变化,免疫组织化学检测CD105的表达,以及应用激光共聚焦显微镜下脉络膜血管平铺法测量各组CNV面积.结果 FFA显示实验组严重渗漏发生率明显低于生理盐水组和空白对照组;实验组光镜下光凝部位新生血管和内皮细胞数明显低于对照组;CD105的表达实验组明显低于生理盐水组和空白对照组;激光共聚焦显微镜下CNV定量分析显示实验组CNV面积(17352±1321)μm2小于生理盐水组(23043±1424)μm2和空白对照组(25937±1744)μm2,其差异具有显著统计学意义(F=342.4,P＜0.001).结论 重组人血管内皮抑素(恩度)可以有效抑制动物模型的CNV形成,为治疗CNV提供了一种新的可能途径.     

小鼠脉络膜血管新生模型中FLK-1的表达

目的:探讨小鼠脉络膜血管新生(choroidal neovascu larization, CNV)模型中FLK-1的表达.方法:采用激光击射的方法诱导成年C57BL/6小鼠CNV模型,术后行脉络膜灌流铺片及组织病理学检查观察CNV的发展.应用免疫组织化学染色探讨正常视网膜以及激光术后10d CNV模型中FLK-1的表达.结果:术后10d所有激光光斑均发展为实验性CNV,其中血管内皮细胞,类视网膜色素上皮细胞(retinal pigmented epithelium,RPE)及类成纤维细胞FLK-1表达强烈;在正常视网膜中,FLK-1仅在RPE、内核层及神经节细胞层有微弱的表达.结论:FLK-1参与了实验性CNV的发病过程,提示拮抗FLK-1可能成为防治CNV有效的生物学方法之一.     

Expression of FLK-1 in laser-induced choroidal neovascularization in mouse

AIM: To investigate the expression of vascular endothelial growth factor receptor 2 (VEGFR-2, also known as FLK-1) in laser-induced choroidal neovascularization (CNV) in mouse. METHODS: CNV was induced in C57BL/6 mouse eyes by krypton laser photocoagulation. Choroidal fluorescein angiography and histopathological examination were used to assess the development of experimental CNV. Cryostat sections from lesions on day 10 after laser treatment and normal eyes were prepared for immunohistochemistry for FLK-1. RESULTS: Laser-induced CNV developed in all lesions on day 10. The expression of FLK-1 was detected in endothelial cells, retinal pigmented epithelium (RPE)-like cells and fibroblast-like cells in neovascular lesions. In normal adult mouse retinas, FLK-1 expression was mainly observed in RPE cells, inner nuclear and ganglion cell layers. CONCLUSION: Our findings demonstrate that expression of FLK-1 may play a role in the formation of laser-induced CNV in mice, which suggests that FLK-1 may be a promising potential target for antiangiogenesis therapy for CNV.