Macrophage depletion diminishes lesion size and severity in experimental choroidal neovascularization

PURPOSE: Macrophage recruitment to the choroid has been proposed to contribute to the pathogenesis of choroidal neovascularization (CNV) in AMD. The study was conducted to determine whether treatment with clodronate liposomes (CL(2)MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS: Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL(2)MDP-lip intraperitoneally and subcutaneously 72 and 24 hours before and every 2 to 3 days after laser injury. Control mice received injections of either PBS alone or PBS liposomes. Blood monocyte and choroidal macrophage depletion were documented by flow cytometry and choroidal flatmount preparation analysis, respectively. Two weeks after laser injury, mice were injected intravenously with fluoresceinated dextran. The right eyes were removed and prepared for flatmount analysis of CNV surface area (in relative disc areas or DA), vascularity (relative fluorescence), and cellularity (propidium iodide stain). The mice were then perfused with 10% formaldehyde, and the left eyes were removed for histopathology. The means of the various parameters for four CNV lesions per eye were calculated. Fluorescein angiography was also performed. RESULTS: Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL(2)MDP-lip-treated mice. Compared with the control, flatmount analysis of macrophage depleted mice demonstrated a significant reduction in size of the CNV area (2.8 +/- 0.5 DA vs. 1.4 +/- 0.1 DA; P < 0.043). The treated group also revealed less vascularity (1.6 +/- 0.1 units vs. 1.1 +/- 0.0 units; P < 0.0092) and cellularity of CNV lesions (3.3 +/- 0.6 DA vs. 1.7 +/- 0.1 DA, P < 0.04). Histopathology revealed that, in the macrophage-depleted group, CNV was smaller in diameter (1270 +/- 73 pixels vs. 770 +/- 82 pixels, P < 0.0006) and thickness (120 +/- 7 pixels vs. 96 +/- 7 pixels, P < 0.019). CONCLUSIONS: Macrophage depletion using CL(2)MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.     

激肽释放酶结合蛋白对脉络膜新生血管抑制作用研究

目的 观察激肽释放酶结合蛋白(KBP)对脉络膜新生血管(CNV)的抑制作用.方法 40只Brown Norway大鼠随机分为KBP组和对照组,每组均为20只,右眼为实验眼.采用532 nm激光建立CNV模型.激光光凝后1周行荧光素眼底血管造影(FFA)检查.检查后第2天,KBP组大鼠玻璃体腔注射浓度为4 mg/ml的KBP溶液5μl,对照组大鼠玻璃体腔注射5μl去离子水.两组大鼠注射后1、2、3周行FFA检查,各时间点检查后,分别摘除5只大鼠眼球制作石蜡切片进行苏木精-伊红染色、血管内皮生长因子(VEGF)和色素上皮衍生因子(PEDF)免疫组织化学3-氨基-9-乙基咔唑染色.应用Image Pro Plus6.0图像分析系统,测量CNV渗漏面积和激光光斑区累积吸光度[A,旧称光密度(OD)]值.结果 激光光凝后1周,FFA检查结果显示CNV形成,可见荧光渗漏.注射后1、2、3周,KBP组大鼠CNV渗漏逐渐减少;对照组大鼠CNV渗漏随观察时间延长而增加.光学显微镜观察发现,KBP组光凝斑面积较对照组光凝斑面积逐渐缩小,新生血管亦逐渐萎缩;对照组光凝斑处新生血管持续存在.免疫组织化学检查结果显示,两组大鼠激光光凝后1周,视网膜VEGF(F=1.29)、PEDF(F=6.29)表达平均累积A值比较,差异均无统计学意义(P＞0.05).注射后1、2、3周,两组大鼠视网膜VEGF、PEDF表达平均累积A值比较,差异均有统计学意义(VEGF:F=14.16、66.89、24.34,PEDF:F=4.22、62.04、233.05;P＜0.001).结论 玻璃体腔注射KBP能有效抑制CNV的形成和发展.     

Macrophage depletion inhibits leukocyte recruitment in experimental melanin-induced uveitis (EMIU)

Background. To study the role of macrophages in experimental melanin-induced uveitis (EMIU), we used the method of intravital microscopy to analyse changes in leukocyte adhesion to iris venules of live rats with EMIU after pretreatment with liposomal clodronate. Materials and methods. EMIU was induced in Lewis rats (n=48) by intraperitoneal immunisation with bovine crude melanosomes emulsified in complete Freund's adjuvant (CFA) and pertussis toxin (PTX). Control animals received CFA and PTX only (n=12) or no injection (n=6). Animals were treated with liposomal clodronate (DMDP-lip) or empty liposomes on days ?2, +1, 4, 6 and 8. Using IVM, postcapillary iris venules of rats were examined to quantify leukocyte rolling and firm adhesion to the vascular endothelium. Results. Depletion of macrophages caused a decreased percentage of rolling leukocytes on day 8 (2±1.1% vs 15.2±1.6%, DMDP-lip vs EMIU, mean ±SEM, ANOVA, p<0.05) and day 10 (2.6±0.3% vs 14.2±1.6%). A signi-ficant decline in the number of firmly adherent leukocytes was detected on days 8 and 10 (88±13/mm² vs 175±18/mm² and 129±13/mm² vs 372±31/mm², DMDP-lip vs EMIU). Treatment with empty liposomes showed no changes in leukocyte firm adhesion. Conclusions. Elimination of macrophages prevents the induction of EMIU. In autoimmune-mediated uveitis, macrophages play a crucial role in the initiation of leukocyte-endothelium interaction.     

Multimodal imaging of experimental choroidal neovascularization

AIM: To compare choroidal neovascularization (CNV) lesion measurements obtained by in vivo imaging modalities, with whole mount histological preparations stained with isolectin GS-IB4, using a murine laser-induced CNV model. METHODS: B6N.Cg-Tg(Csf1r-EGFP)1Hume/J heterozygous adult mice were subjected to laser-induced CNV and were monitored by fluorescein angiography (FA), multicolor (MC) fundus imaging and optical coherence tomography angiography (OCTA) at day 14 after CNV induction. Choroidal-retinal pigment epithelium (RPE) whole mounts were prepared at the end of the experiment and were stained with isolectin GS-IB4. CNV areas were measured in all different imaging modalities at day 14 after CNV from three independent raters and were compared to choroidal-RPE whole mounts. Intraclass correlation coefficient (ICC) type 2 (2-way random model) and its 95% confidence intervals (CI) were calculated to measure the correlation between different raters'' measurements. Spearman''s rank correlation coefficient (Spearman''s r) was calculated for the comparison between FA, MC and OCTA data and histology data. RESULTS: FA (early and late) and MC correlates well with the CNV measurements ex vivo with FA having slightly better correlation than MC (FA early Spearman''s r=0.7642, FA late Spearman''s r=0.7097, and MC Spearman''s r=0.7418), while the interobserver reliability was good for both techniques (FA early ICC=0.976, FA late ICC=0.964, and MC ICC=0.846). In contrast, OCTA showed a poor correlation with ex vivo measurements (Spearman''s r=0.05716) and high variability between different raters (ICC=0.603). CONCLUSION: This study suggests that FA and MC imaging could be used for the evaluation of CNV areas in vivo while caution must be taken and comparison studies should be performed when OCTA is employed as a CNV monitoring tool in small rodents.     

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.     

Electron microscopic features of experimental choroidal neovascularization

We produced choroidal neovascularization in the rhesus monkey by diminishing the blood supply to the inner retina and producing defects in Bruch's membrane by photocoagulation. The neovascular fronds which developed either infiltrated the subretinal space or proliferated through necrotic and gliotic retina into the vitreous cavity. Sequential electron microscopic sections of neovascular fronds in the subretinal space demonstrated that the advancing capillary sprouts were composed of primitive endothelial tubes surrounded by pericytes and enmeshed in a loose basement-membrane-like substance. More mature capillaris and displayed endothelial fenestrations and endothelial-pericyte membranous contacts. Large neovascular fronds developed major feeding vessels that closely resembled normal small choroidal arteries and veins. Retinal pigment epithelial cells in various guises were in constant association with proliferating neovascular networks.     

Ketorolac inhibits choroidal neovascularization by suppression of retinal VEGF

We assessed the effect of topical ketorolac on laser-induced choroidal neovascularization (CNV), measured retinal PGE₂ and VEGF levels after laser treatment, and determined the effect of ketorolac on PGE₂ and VEGF production. Six laser burns were placed in eyes of rats which then received topical ketorolac 0.4% or artificial tears four times daily until sacrifice. Fluorescein angiography (FA) was performed at 2 and 3 weeks and retinal pigment epithelium-choroid-sclera flat mounts were prepared. The retina and vitreous were isolated at 1, 3, 5, 7, and 14 days after laser treatment and tested for VEGF and PGE₂. Additional animals were lasered and treated with topical ketorolac or artificial tears and tested at 3 and 7 days for retinal and vitreous VEGF and PGE_2. Ketorolac reduced CNV on FA by 27% at 2 weeks (P < 0.001) and 25% at 3 weeks (P < 0.001). Baseline retina and vitreous PGE₂ levels were 29.4 μg/g and 16.5 μg/g respectively, and reached 51.2 μg/g and 26.9 μg/g respectively, 24 h after laser treatment (P < 0.05). Retinal VEGF level was 781 pg/g 24 h after laser treatment and reached 931 pg/g by 7 days (P < 0.01). Ketorolac reduced retinal PGE₂ by 35% at 3 days (P < 0.05) and 29% at 7 days (P < 0.001) and retinal VEGF by 31% at 3 days (P = 0.10) and 19% at 7 days (P < 0.001). Topical ketorolac inhibited CNV and suppressed retinal PGE₂ and VEGF production.     

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM: To determine the effects of intravitreal resveratrol (RSV) on murine laser-induced choroidal neovascularization (CNV). METHODS: The toxicity of RSV to choroidal endothelial cell (CEC) was measured using thiazolyl blue tetrazolium bromide (MTT) assay. Effects of RSV on choroidal endothelial cell (CEC) migration were evaluated with a modified Boyden chamber assay, while tube formation was evaluated in a 2-D gel assay. CNV was induced by laser photocoagulation in mice. The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography (FA), confocal analysis of isolectin B4 labeled choroidal flat mounts, and histologic examination of CNV membranes. Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). RESULTS: No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 μmol/L (P>0.05). RSV inhibited vascular endothelial growth factor (VEGF)-induced CEC migration (P<0.05) and tube formation (P<0.05) in vitro. Furthermore, intravitreal injection of RSV significantly inhibited laser induced CNV formation in mice. The FA leakage, CNV volume and CNV area analysis revealed that there were 41%, 45%, and 58% reduction in RSV-treated eyes (1.691±0.1032, 178 163±78 623 μm3 and 6508±619.0 μm2, respectively) compared with those in control (2.724±0.08447, 379 676±98 382 μm3 and 16 576±2646 μm2, respectively; P<0.05). Phospho-VEGFR2 expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control (P<0.05). CONCLUSION: Intravitreal injection of RSV exerts an inhibitory effect on CNV, which may through suppressing endothelial cell migration, tube formation and VEGFR2 phosphorylation.     

康柏西普对实验性脉络膜新生血管的作用机制研究

目的　探究康柏西普对实验性脉络膜新生血管（choroidal neovascularization，CNV）的作用机制。方法　选取40只7周龄的雄性BN大鼠，采用多波长氪激光对豚鼠左眼行视网膜光凝，制作CNV大鼠模型，并随机分为4组，空白对照组、模型组、低剂量康柏西普组和高剂量康柏西普组；于造模后7 d、14 d注射康柏西普，并在造模后21 d进行右眼眼底彩照、眼底荧光素血管造影（fundus fluorescein angiography，FFA）检查，检测CNV 发生率及渗漏面积，使用FITC灌注脉络膜铺片测量CNV面积，使用HE染色观察大鼠视网膜结构，使用Western blot检测大鼠视网膜中血管内皮生长因子（vascular endothelial growth factor，VEGF）、缺氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）、丝氨酸/苏氨酸激酶 （serine/threonine kinase，AKT）表达情况。结果　除吲哚菁绿血管造影荧光点数、CNV发生率外，相比空白对照组，模型组大鼠CNV渗透面积，荧光素渗漏面积，脉络膜结构损伤程度，视网膜中 HIF-1α、AKT和VEGF蛋白的表达均显著升高，差异均有统计学意义（均为P<0.05）；相比模型组，康柏西普组大鼠上述各指标显著降低，高剂量康柏西普组上述各指标显著低于低剂量康柏西普组，差异均有统计学意义（均为P<0.05）。结论　康柏西普可以抑制脉络膜视网膜中 HIF-1α、AKT和VEGF蛋白的表达，从而抑制由氪激光光凝眼底而产生的实验性CNV生长。     

A novel imaging technique for experimental choroidal neovascularization

PURPOSE: Choroidal neovascularization (CNV) is the end point of several ocular diseases that lead to blindness. The authors developed an imaging technique for visualizing and quantifying morphologic changes associated with experimental laser-induced CNV. METHODS: CNV was induced using laser energy to disrupt Bruch's membrane. Rats were euthanatized immediately after laser injury and at 1, 2, 3, 4, 7, 14, and 60 days. Nonlasered eyes were used as the control. Eyes were enucleated and fixed, and the posterior eye cups were fluorescently labeled with markers for nuclei (DAPI; 4',6'-diamino-2-phenylindole), endothelial cells (isolectin IB4), microglia (CD11b), and filamentous actin (phalloidin). FITC-dextran perfusion was compared with our technique. A confocal microscope was used to evaluate flatmounted specimens. Computer software generated three-dimensional reconstructions for qualitative and quantitative analysis of confocal image stacks. RESULTS: In nonlasered areas, RPE cells were visualized as a uniform hexagonal array. Immediately after laser exposure, a circular area devoid of fluorescent labeling was observed, indicating disruption of the choroid-Bruch's membrane-RPE complex. One day after laser exposure, cellular debris and fragmented nuclei were present, and an autofluorescent ring was visible at the site of Bruch's membrane disruption. The ring correlated with bubble formation and CNV induction. Three days after laser injury, phalloidin-labeled RPE cells and isolectin-labeled endothelial cells increased significantly, reflecting cell proliferation and migration. By day 4, isolectin-positive cells forming vascular tubes were visualized. The volume of CNV vessels increased exponentially during the next 3 days. By 7 days, a well-defined isolectin-labeled CNV network was present, and its volume was preserved for several weeks. CNV volumes calculated on the basis of FITC-dextran perfusion were significantly lower than volumes obtained using lectin-labeled samples. CONCLUSIONS: A novel imaging technique was developed that allows a three-dimensional reconstruction and measurement of laser-induced CNV lesions in rat choroid/RPE flatmounts. This technique provides excellent morphologic detail and facilitates the study of critical early events in CNV, including the rupture of Bruch's membrane and the formation of endothelial clusters before vessel formation. CNV complexes are labeled at an earlier stage and more reproducibly than with FITC-dextran perfusion, providing a more accurate preclinical evaluation of antiangiogenic molecules.     

Soluble EphB4 regulates choroidal endothelial cell function and inhibits laser-induced choroidal neovascularization

PURPOSE: The purpose of this study was to evaluate the effect of a soluble monomeric form of the EphB4 extracellular domain (sEphB4) on choroidal endothelial cell (CEC) migration and tube formation and on experimental laser-induced choroidal neovascularization (CNV). METHODS: EphrinB2 and EphB4 expression in CECs was investigated by Western blot analysis and immunohistochemistry. Effects of sEphB4 (0.5-3 microg/mL) on CEC migration were evaluated with a modified Boyden chamber assay. Tube formation was assayed in CEC cultures in collagen gel. CNV was induced in rats by laser photocoagulation. The effects of intravitreal injection of sEphB4 on CNV development were evaluated at day 14 by fluorescein angiography (FA), confocal volumetric analysis of isolectin-B4 labeled flatmounts, and histologic examination of CNV membranes. RESULTS: CEC cells express both EphB4 and EphrinB2, according to Western blot analysis. Immunohistochemical sections of rat eye showed immunoreactivity for both EphB4 and EphrinB2 in the choroidal endothelium. sEphB4 reduced CEC migration in response to vascular endothelial growth factor (P < 0.01). Similarly, sEphB4 inhibited CEC tube formation in a dose-dependent manner. EphB4, and to a lesser extent EphrinB2, were detected on vascular channels within laser-induced CNV membranes. Intravitreal injection of sEphB4 inhibited laser-induced CNV formation. CNV membranes showed a reduction in leakage score (P < 0.05), and membrane volumes were reduced in size (P < 0.05). Histologic analysis revealed that vascularity was reduced in sEphB4-treated membranes. CONCLUSIONS: Recombinant soluble monomeric EphB4 exerts an inhibitory effect on choroidal angiogenesis in vitro and in vivo. It should be further evaluated for its potential as a novel therapy for CNV.     

AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization

PURPOSE: Adeno-associated viral (AAV) vectors have been used to express several different proteins in the eye. The purpose of this study was to determine whether AAV-mediated intraocular gene transfer of pigment epithelium-derived factor (PEDF) inhibits the development of choroidal neovascularization (CNV) in a murine model. METHODS: C57BL/6 mice were given intravitreous or subretinal injections of a PEDF expression construct packaged in an AAV vector (AAV-chicken beta-actin promoter-exon 1-intron 1[CBA]-PEDF) or control vector (AAV-CBA-green fluorescent protein[GFP]). After 4 or 6 weeks, the Bruch's membrane was ruptured by laser photocoagulation at three sites in each eye. After 14 days, the area of CNV at each rupture site was measured by image analysis. Intraocular levels of PEDF were measured by enzyme-linked immunosorbent assay. RESULTS: Four to six weeks after intraocular injection of AAV-CBA-PEDF, levels of PEDF in whole-eye homogenates were 6 to 70 ng. The average area of CNV at sites of the Bruch's membrane rupture showed no significant difference in eyes injected with AAV-CBA-PEDF compared with uninjected eyes. In contrast, 4 to 6 weeks after intraocular injection of 1.5 x 10(9) or 2.0 x 10(10) particles of AAV-CBA-PEDF, the area of CNV at the Bruch's membrane rupture sites had significantly decreased compared with CNV area at rupture sites in eyes injected with AAV-CBA-GFP. CONCLUSIONS: These data suggest that intraocular expression of PEDF or other antiangiogenic proteins with AAV vectors may provide a new treatment approach for ocular neovascularization.     

Expression of pigment epithelium-derived factor in experimental choroidal neovascularization

PURPOSE: To investigate the expression of pigment epithelium-derived factor (PEDF) in the rat laser-injury model of choroidal neovascularization (CNV). METHODS: Retinas were immunostained for PEDF at different times (1, 2, and 3 weeks) after laser injury. Levels of PEDF protein in the vitreous at 1, 3, 7, 14, and 28 days after laser injury were also assayed by Western blot. RESULTS: Protein levels of PEDF in the vitreous were increased during the first 7 days after CNV induction. Immunostaining for PEDF was observed throughout normal nonlasered control retinas, sham-lasered retinas, and areas remote to laser lesions, which were generally more intense in the outer nuclear layer (ONL) and less intense in the internal nuclear layer (INL). Decreased expression of PEDF was observed in flanking areas adjacent to the injury site and was confined mainly to the ONL. In the injury sites, immunostaining within the ONL was either absent or decreased for up to 3 weeks after laser injury (the duration of the study). Preadsorption of the anti-PEDF antibody with the immunizing peptide blocked specific labeling in the retina. CONCLUSIONS: These results demonstrate an inverse correlation of expression of PEDF and formation of CNV in the experimental model and suggest that decreased expression of PEDF plays a permissive role in the formation of CNV. PEDF analogues may be a reasonable treatment strategy for CNV.     

Photodynamic therapy of experimental choroidal neovascularization in the mouse

PURPOSE: To evaluate the qualitative and quantitative effects of verteporfin photodynamic therapy (PDT) on laser-induced choroidal neovascularization (CNV) in the mouse. METHODS: PDT was applied to the normal mouse fundus using light doses of 32, 64, and 83 s, and histological analysis of the treated areas was performed. CNV was induced using krypton laser photocoagulation of the fundus, and the CNV lesions were subsequently treated with PDT using light doses of 32, 64, and 83 s. Enucleated eyes were analyzed with light and transmission electron microscopies, and measurements of CNV size were done on histologic sections and on isolectin B4-stained choroidal flat mounts. RESULTS: PDT induced a light dose-dependent damage to the surrounding neural retina in normal eyes. At a light dose of 32 s, minimal damage was detected in the neural retina, whereas higher light doses caused distortion and disruption of the outer and inner nuclear layers and of the retinal pigment epithelium. When PDT was applied over laser-induced CNV lesions, the relative height of the lesions was significantly reduced (p < 0.05) using all light doses. Transmission electron microscopy 1 day after PDT treatment revealed occlusion of many of the CNV vessels. One week after PDT treatment, the CNV lesions contained patent vessels irrespective of light dose applied. Accordingly, PDT treatment inhibited (p < 0.05) but did not halt CNV lesion growth. CONCLUSIONS: PDT treatment of laser-induced CNV may create an acute occlusion of neovessels and an inhibition of CNV lesion growth without apparent injury to the surrounding neural retina. However, PDT-treated areas will remain vascularized with continued growth of the CNV lesion, which in turn may explain the often limited effect of PDT in patients with neovascular age-related macular degeneration. Elevating the PDT light dose will not increase the treatment effect substantially but may lead to increased collateral injury.     

Bone marrow-derived progenitor cells contribute to experimental choroidal neovascularization

PURPOSE: The pathogenesis of choroidal neovascularization (CNV) is postulated to be driven by angiogenesis, a process in which the cellular components of the new vessel complex are derived from cells resident within an adjacent preexisting capillary. Recently, an alternative paradigm, termed postnatal vasculogenesis, has been shown to contribute to some forms of neovascularization. In vasculogenesis, the cellular components of the new vessel complex are derived from circulating vascular progenitors from bone marrow. In the current study, transplantation of green fluorescent protein (GFP)-labeled bone marrow and laser-induced CNV were combined to examine the contribution of vasculogenesis to the formation of CNV. METHODS: Ten adult C57BL/6 female mice were used as recipients for bone marrow transplantation. Bone marrow was obtained from three C57BL/6 female mice transgenic for the beta-actin promoter GFP. One month after bone marrow transplantation, CNV was induced in recipient mice by making four separate burns in the choroid of each eye with a red diode laser. Four weeks after CNV was induced, eyes of recipient mice were processed for immunohistochemistry to detect GFP and markers for vascular smooth muscle cells (alpha-smooth muscle actin, desmin, and NG2 chondroitin sulfate proteoglycan), endothelial cells (CD31, BS-1 lectin), or macrophages (F4/80). RESULTS: GFP-labeled cells represented 17% of the total cell population in the lesion. Many of the GFP-labeled cells were immunoreactive for alpha-smooth muscle actin (39%), desmin, NG2, CD31 (41%), BS-1 lectin, or F4/80. GFP-labeled cells were morphologically indistinguishable from cells normally present in CNV lesions. CONCLUSIONS: This study is the first to demonstrate that bone marrow-derived progenitor cells are a source of endothelial and smooth musclelike cells in CNV.     

荧光抗体标记法在小鼠脉络膜新生血管中的应用

背景脉络膜新生血管（CNV）是导致多种眼底疾病视力损害的主要原因,准确建立CNV模型对于其实验和临床研究具有重要的意义,但目前尚无一种可重复且可靠的评估CNV模型及其有效治疗的方法。目的探讨荧光抗体标记法在定性和定量评价氪激光诱导的小鼠CNV中的应用价值。方法选取15只雄性SPF级C57BL／6J小鼠,用氪激光（波长为647．1am）视网膜光凝的方式建立CNV模型。分别于光凝后5rain,4、7、14及28d随机选取3只模型小鼠行眼底照相和荧光素眼底血管造影（FFA）检查。摘除眼球行脉络膜铺片,用荧光抗体（DAPI、isolectin-B4及phalloidin）分别标记光凝区域的细胞核、内皮细胞和肌动蛋白。用Imageproplus6．0软件测量CNV面积。结果FFA和脉络膜铺片检查结果显示激光光凝后5min及4d无CNV形成,光凝后7d开始出现CNV。光凝后7、14、28d有荧光素渗漏的光凝斑的百分率分别为76．47％（26／34）、81．81％（18／22）和50．00％（5／10）。脉络膜铺片后荧光显微镜检测结果显示,在正常的未光凝区域,视网膜色素上皮（RPE）细胞呈均匀一致的六边形排列;光凝后5min,在光凝部位可检测到一个环形荧光素缺损区,表明脉络膜-Bruch膜-RPE复合体已被破坏;光凝后4d出现一些细胞碎片以及核碎片,Bruch膜破损处可见自发荧光环。光凝后7d时,激光损伤区出现界限清楚的CNV网,并持续到28d。7、14及28d时根据脉络膜铺片所测得的CNV面积分别为（7．99±0．42）×10^3、（16．89±8．77）×10^3、（14．37±4．02）×10^3μm^2,差异有统计学意义（F=17．340,P=0．000）。光凝后14d和28d脉络膜铺片上CNV面积相差不大,但与7d时相比面积均明显增加（q=16．46、q=15．54,P〈0．01）。结论荧光抗体标记法不仅能很好地显示氪激光诱导的小鼠实验性CNV及其形态,而且能测定CNV的面积,为抗新生血管药物应用的疗效研究提供依据。     

沙利度胺治疗脉络膜新生血管的实验研究

目的探讨沙利度胺（thalidomide）对脉络膜新生血管（CNV）的抑制作用及毒副作用。方法健康C57BL／6J小鼠采用氪红激光光凝建立CNV模型。随机分组：A组为对照组;B组腹腔注射沙利度胺,浓度为10mg／kg;C组腹腔注射沙利度胺浓度为50mg／kg。连续应用10d。记录实验前后小鼠体重变化。从防御反射、捕捉难易度、进食量、皮毛颜色及精神状态等5个方面来评估小鼠行为学改变。病理组织学检查测量CNV组织厚度。结果小鼠体重变化有明显差异,C组小鼠体重增加明显小于A组和B组（P〈0．01）。A组和B组小鼠行为学基本没有改变。C组小鼠行为学改变明显。3组小鼠CNV组织中央厚度有明显差异;A组小鼠CNV组织中央厚度高于B、C两组（P〈0．01）。结论沙利度胺能够抑制实验性CNV;在常规用药剂量下,小鼠能较好的耐受沙利度胺,当药物浓度过大时,其抑制CNV的作用没有增加,但副作用明显严重。