Photodynamic therapy with verteporfin in a rabbit model of corneal neovascularization

PURPOSE: To determine the efficacy of photodynamic therapy (PDT) with verteporfin (Visudyne; Novartis AG, Basel, Switzerland) for treatment of corneal neovascularization in a rabbit eye model. METHODS: Corneal neovascularization was induced in Dutch belted rabbits by placing an intrastromal silk suture near the limbus. Verteporfin was administered by intravenous injection at a dose of 1.5 mg/kg, and the pharmacokinetics of verteporfin distribution in the anterior segment or PDT-induced (laser energy levels 17, 50, and 150 J/cm(2)) regression of corneal blood vessels were then determined. To assess PDT-induced toxicity of the anterior segment, corneal and iris/ciliary body histology, and IOP were evaluated after PDT. RESULTS: Verteporfin accumulation in vascularized regions of the cornea and the iris/ciliary body tissue were time dependent and maximum levels achieved at 60 minutes after injection. In rabbits, PDT of corneal vessels using laser energy of 17 or 50 J/cm(2) resulted in 30% to 50% regression of corneal neovascularization; however, in these animals, a rapid regrowth of new blood vessels occurred between 3 and 5 days. In the rabbits receiving PDT using laser energies of 150 J/cm(2), the mean vessel regression was 56%. During the nine days of the laser therapy follow-up period, no vessel regrowth was observed in these rabbits. Histologic examination of the anterior segment after PDT (150 J/cm(2)) showed localized degeneration of the corneal blood vessels without observable change in other anterior segment structures. CONCLUSIONS: These results provide evidence that PDT can produce significant regression of neovascular corneal vessels with no observable toxicity to the anterior segments. However, the optimal laser energy necessary to induce long-term regression (150 J/cm(2)) was three times that used to treat choroidal neovascularization.     

Age-related changes in murine limbal lymphatic vessels and corneal lymphangiogenesis

Important risk factors for graft rejection after corneal transplantation are pathologic corneal lymphangiogenesis and young recipient age. Purpose of this study was to investigate whether there are age-related differences in normal murine limbal and pathologic corneal lymphatic vessels, which could partly explain the unequal outcome of corneal transplantation in young versus old recipients. Furthermore, we investigated whether these observed differences correlate with changes in allograft survival in the murine model of corneal transplantation. Corneal whole mounts from untreated young (aged 6-8 weeks), untreated old (aged 9-15 months) and young and old mice after suture-induced, inflammatory corneal neovascularization were prepared and stained with LYVE-1 as a lymphendothelial marker. Angles of corneal parts with and without a main circumferential limbal lymphatic vessel were measured and then related to the total 360° of corneal circumference. Centrally directed vascular extensions from the main limbal lymphatic vessel (“sprouts”) of previously untreated old mice were counted. Concerning the outgrowth of pathologic lymphatic vessels after inflammatory corneal neovascularization, the area covered with pathologic lymphatic vessels was detected by an algorithm on digitized whole mounts using cell^F^® software. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed with one recipient group being young, the other group being old mice. In young, untreated mice, 70.5% of the total corneal circumference was covered by a main circumferential limbal lymphatic vessel versus 60.8% in old, untreated mice. Comparing the number of centripedal vascular extensions from the main limbal lymphatic vessel (“sprouts”), untreated old mice had significantly less extensions than young, untreated mice (p < 0.001). After an inflammatory stimulus, old mice had significantly less pathologic corneal lymphatic vessels than young mice (42% less, p < 0.001). Comparing the survival proportions after corneal transplantation, old recipient mice showed a significantly better graft survival 6 weeks after transplantation (65% versus 33%, p < 0.05). Thus, limbal lymphatic vascular sprouts and inflammation-induced pathologic corneal lymphangiogenesis decrease with age. The lower lymphangiogenic potency of older mice may explain the better outcome of corneal transplantations in old recipients, supporting the concept that lymphangiogenesis is an important risk-factor for corneal transplant rejection.     

Inhibition of corneal neovascularization by genetic ablation of CCR2

PURPOSE: To determine if genetic ablation of the chemokine receptor CCR2 (involved in leukocyte and endothelial chemotaxis) inhibits the development of corneal neovascularization. METHODS: Wild-type C57BL/6J mice, as well as species-specific counterparts with targeted homozygous disruption of the CCR2, underwent chemical and mechanical denudation of corneal and limbal epithelium. Corneas were harvested 2 weeks after injury. Neovascularization was quantified by CD31 immunostaining. RESULTS: The mean percentages of neovascularized corneal area in control mice and CCR2-deficient mice 2 weeks after denudation were 58.3% and 38.8% (P = 0.047), respectively. CONCLUSIONS: Development of corneal neovascularization is inhibited in CCR2-deficient mice.     

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)     

角膜缘血管网切断联合生物羊膜移植治疗角膜新生血管

目的:探讨角膜缘血管网切断联合生物羊膜移植治疗角膜新生血管的安全性和有效性。方法:对沙眼、热烧伤、感染、外伤等原因所致的角膜新生血管52例52眼行角膜缘血管网切断联合生物羊膜移植术。合并角膜溃疡12眼,合并角膜白斑3眼;新生血管累及角膜中央区有8眼,旁中央区10眼,周边区24眼,角膜缘区10眼。所有患者术后随访6mo,观察眼部自觉症状、视力及并发症。结果:术后视力提高>1行者44眼,术后视力未见变化者8眼。原有新生血管数量减少,角膜水肿混浊程度减轻,炎症反应得到控制、角膜透明性有所提高。术后6mo,新生血管完全消失22眼(42%);复发17眼(33%)。结论:角膜缘血管网切断联合生物羊膜移植治疗角膜新生血管有效,无明显副作用,能减轻急性期的炎症反应,缩短病程。     

Mouse strain-dependent heterogeneity of resting limbal vasculature

PURPOSE: Heterogeneity of the extent of angiogenesis induced by exogenous growth factors may be determined by genetic influences. Because angiogenesis is the formation of new vessels from preexisting ones, strain-related influences on na?ve resting limbal vessel phenotype and gene expression were determined in mice having divergently low and high angiogenic responses. METHODS: Resting limbal vessel surface area and density and extent of bFGF-induced corneal angiogenesis were determined in C57BL/6J, BALB/cJ, F1 intercross identical with C57BL/6J X 129S3/SvIM, and 129S3/SvIM mouse strains by quantitative three-dimensional reconstruction confocal microscopy. Strain-related influences on pro- and antiangiogenic gene expression in na?ve cornea were determined by quantitative real-time RT-PCR. RESULTS: The strain-dependent rank order of resting limbal vessel surface area and resting vessel density paralleled bFGF-induced neovascularization: 129S3/SvIM > BALB/cJ, F1 > C57BL/6J (P < 0.0006). Pigment epithelium-derived factor (PEDF) was increased more than 67-fold compared to Ang-2 in resting cornea of both C57BL/6J and 129S3/SvIM strains (P < 0.0001; P < 0.0001), suggesting a strongly antiangiogenic environment. The corneas of the C57BL/6J mice demonstrated 1.8-, 1.5-, and 1.7-fold increased mRNA levels for Flt-1, VEGF, and bFGF, respectively (P < 0.02; P < 0.04; P < 0.02); however, TSP-1 expression was increased 2.4-fold compared with 129S3/SvIM (P < 0.0004). CONCLUSIONS: Strain-dependent differences in the resting limbal vessel surface area and density correlated with heterogeneity in the extent of bFGF-induced angiogenesis. Differences in pro- and antiangiogenic gene expression levels in resting cornea may influence vascular limbal phenotype during quiescence and may predict susceptibility to angiogenesis-dependent diseases.     

Effect of photodynamic therapy with ATX-S 10 (Na) on experimental choroidal neovascularization

PURPOSE: To evaluate an appropriate irradiative condition for selective occlusion of experimental choroidal neovascularization(CNV) with photodynamic therapy (PDT) using ATX-S 10 (Na). METHODS: Experimental CNV was induced in monkey eyes by laser photocoagulation. PDT(dose of irradiative energy 40 to 80J/cm2) was performed after 3.5 mg/kg of body weight intravenous injections of ATX-S 10(Na). CNV and retinal vessel occlusion induced by PDT was evaluated by fluorescein angiography (FA) at 1 and 7 days after irradiation. If FA showed no fluorescein dye leakage from CNV at 1 and 7 days after irradiation, CNV was evaluated by histopathological analysis at 7 days after irradiation. RESULTS: Within 30 to 33 minutes after ATX-S 10(Na) injection and irradiation with 50 to 60 J/ cm2, FA showed no fluorescein dye leakage from CNV and no closure of retinal vessels at 1 and 7 days after irradiation. Light micrographs showed occluded CNV, and retinal vessels remained patent and there was no apparent change in the inner layer of the retina. CONCLUSIONS: Irradiative condition of ATX-S10 (Na) 3.5 mg/kg was appropriate 30 to 33 minutes after ATX-S 10(Na) injection and irradiation with 50 to 60 J/cm2.     

Sustained inhibition of corneal neovascularization by genetic ablation of CCR5

PURPOSE: To determine whether genetic ablation of the CC chemokine receptor CCR5 (involved in leukocyte and endothelial chemotaxis) inhibits the development of corneal neovascularization. METHODS: Wild-type C57BL/6J mice and species-specific counterparts with targeted homozygous disruption of the CCR5 gene underwent chemical and mechanical denudation of corneal and limbal epithelium. Corneas were harvested 2 and 4 weeks after injury. Neovascularization was quantified by CD31 immunostaining. Expression of VEGF protein was quantified by ELISA. RESULTS: The mean percentages of neovascularized corneal area in control mice and CCR5-deficient mice 2 weeks after denudation were 58.3% and 38.5% (P = 0.05), respectively. At 4 weeks after denudation, the corresponding percentages were 67.6% and 44.0% (P = 0.028). In CCR5-deficient mice, VEGF protein levels were reduced 51.1% at 2 weeks (P = 0.05) after injury and 37.3% at 4 weeks (P = 0.03). CONCLUSIONS: CCR5-deficient mice showed a persistent 34% to 35% inhibition of corneal neovascularization for up to 4 weeks. This inhibition correlates with reduced expression of VEGF. These data implicate CCR5 as one essential component in the development of corneal neovascularization.     

The role of PDGF receptor inhibitors and PI3-kinase signaling in the pathogenesis of corneal neovascularization

PURPOSE: Corneal neovascularization remains an unsolved therapeutic problem. Platelet-derived growth factor (PDGF) is directly linked to vessel formation and stabilization. This study was undertaken to elucidate the mechanisms by which PDGF exerts its effects on corneal angiogenesis. METHODS: Corneal neovascularization was induced in C57 mice by removal of the limbal epithelium. When mature vessels appeared after 7 days, mice were treated with the PDGF receptor-beta inhibitor AG 1296 or the phosphatidylinositol 3-kinase (PI3-K)-inhibitors wortmannin and LY294002, respectively, using an intraperitoneally implanted miniosmotic pump. At day 14 after scraping, corneas of treated and untreated (control) mice were dissected and immunostained with FITC-CD31 antibody for endothelial cells and with Cy3-SMA (smooth muscle actin) for pericytes. VEGF (vascular endothelial growth factor), ang1/2 (angiopoietin 1 and 2), and PDGF mRNA levels of treated and untreated corneas were determined by real-time RT-PCR. RESULTS: Mice treated with the PDGF inhibitor AG 1296 showed an inhibition of corneal neovascularization of 21.1% and a reduction of pericytes of 52% in the newly formed vessels compared with untreated animals. VEGF, ang1, ang2, and PDGF mRNA expression was reduced in the corneas of AG 1296-treated mice compared with the respective control. Treatment with the PI3-K inhibitors wortmannin and LY29002 had similar effects, inducing a decrease in corneal neovascularization and a reduction of VEGF, ang1, ang2, and PDGF mRNA levels. CONCLUSIONS: Inhibition of the PDGF signal pathway results in loss of pericytes and a reduction in vessel density in the neovascularized cornea that correlates with reduced expression of PDGF, ang1/2, and VEGF mRNA. Furthermore, PI3-K was shown to be involved in the regulation of VEGF, ang1, and PDGF, as the PI3-K inhibitors wortmannin or LY294002 had similar effects. Because PDGF is a known stimulus for PI3-K activation, it can be postulated that the observed decrease in VEGF, ang1/2, and PDGF mRNA levels on administration of the PDGF inhibitor is caused by the decreased activation of the PI3-K signaling cascade.     

Photodynamic therapy of corneal neovascularization with verteporfin

PURPOSE: To describe the effect of photodynamic therapy (PDT) using verteporfin (Visudyne) on corneal neovascularization (CNV) in two patients. METHODS: Two patients with corneal neovascularization were treated with a nonthermal laser light at 689 nm delivered 15 min after an intravenous infusion of verteporfin. Postoperative outcome of neovascularization was followed clinically (inflammation, intraocular pressure, and visual acuity) and photographically [color photographs and corneal fluorescein and indocyanine green (ICG) angiography] for a minimum of 6 months. RESULTS: Successful photothrombosis of corneal neovascularization was obtained immediately after treatment in the two patients, and regression was verified by corneal fluorescein and ICG angiography. In one case, partial vessel recanalization was observed after 1 month, and treatment was repeated, with complete regression of new vessels. No relevant side effects were observed in our cases. CONCLUSIONS: PDT with verteporfin is an effective and safe procedure indicated for patients with corneal neovascularization; however, multiple sessions may be required.     

光动力学疗法治疗角膜新生血管后角膜的光电镜改变

目的观察应用国产光敏剂行光动力学疗法(PDT)治疗角膜新生血管后角膜的组织学改变。方法碱烧伤有色兔角膜制作角膜新生血管模型。血啉单醚5mg/kg自耳缘静脉注射,不同的能量密度61.2-52.8J/cm2的氲绿激光照射角膜新生血管根部,不注射血啉单醚并单纯行同等能量密度的激光照射组作为对照组,PDT治疗后3h、1周、1个月行角膜光电镜检查。结果PDT后3h可见角膜急性炎症反应,有大量中性粒细胞浸润和少量嗜酸性白细胞浸润,虹膜组织无损伤。PDT后1周角膜炎症反应大部分消失,可见新生血管腔内有无定形物质填塞和许多影子血管。透射电镜显示:PDT组角膜新生血管内皮细胞内线粒体显示出空泡样变,细胞形态不完整。结论血啉单醚作为光敏剂,应用氩绿激光对角膜新生血管行PDT治疗,导致新生血管内皮细胞损伤,能有效封闭角膜新生血管,对周围组织无明显损伤。     

Evaluation of the new photosensitizer Stakel (WST-11) for photodynamic choroidal vessel occlusion in rabbit and rat eyes

PURPOSE: To evaluate the photodynamic potential of a new hydrosoluble photosensitizer (WST-11, Stakel; Steba Biotech, Toussus-Le-Noble, France), for use in occlusion of normal choroidal vessels in the rabbit eye and CNV (choroidal neovascularization) in the rat eye. METHODS: Occlusive and nonocclusive parameters of Stakel and verteporfin photodynamic therapy (PDT) were investigated in pigmented rabbits. Eyes were followed by fluorescein angiography (FA) and histology at various intervals after PDT. RESULTS: When occlusive parameters (fluence of 50 J/cm(2), 5 mg/kg drug dose and DLI [distance to light illumination] of 1 minute) were used, Stakel PDT was efficient immediately after treatment without associated structural damage of the RPE and retina overlying the treated choroid in the rabbit eye. Two days later, total occlusion of the choriocapillaries was seen in 100% of the treated eyes, along with accompanying histologic structural changes in the overlying retina. When the occlusive parameters (fluence, 100 J/cm2; drug dose, 12 mg/m2; and DLI, 5 minutes) of verteporfin PDT were used, occlusion of the choriocapillaries was observed in 89% of the treated eyes. Histology performed immediately after treatment demonstrated structural damage of the overlying retina and RPE layer. Weaker, nonocclusive Stakel PDT parameters (25 J/cm2, 5 mg/kg, and DLI of 10 minutes) did not induce choriocapillary occlusion or retinal lesions on FA or histology. Weaker, nonocclusive verteporfin PDT parameters (10 J/cm2, 0.2 mg/kg, and DLI of 5 minutes) did not induce choriocapillary occlusion. However, histology of these eyes showed the presence of damage in the retinal and choroidal tissues. Moreover, preliminary results indicate that selective CNV occlusion can be achieved with Stakel PDT in the rat eye. CONCLUSIONS: Unlike verteporfin PDT, Stakel PDT does not cause direct damage to the RPE cell layer or retina. These observations indicate that Stakel PDT may have a high potential for beneficial therapeutic outcomes in treatment of AMD.     

兔同种异体角膜缘移植角膜印迹细胞学检测

目的 研究兔角膜缘干细胞缺乏和同种异体角膜缘移植后临床和角膜上皮表型的改变。方法 建立兔角膜缘干细胞缺乏模型,1个月后对治疗组进行同种异体角膜缘移植,术后联合使用免疫抑制剂。比较治疗组与非治疗组的临床表现和角膜表型的改变。结果 兔角膜缘干细胞缺乏后,角膜混浊,新生血管化,持续性上皮缺损;角膜上皮为结膜细胞表型。移植术后角膜上皮完整,新生血管减少,角膜透明度增加;上皮恢复角膜表型。结论 兔同种异体角膜缘移植联合术后使用免疫抑制剂是治疗角膜缘干细胞缺乏症的有效方法。印迹细胞学检查是角膜缘干细胞缺乏症诊断和角膜缘移植术后的评价手段。     

兔眼脉络膜黑色素瘤的第二代光敏剂光动力治疗

目的 观察第二代光敏剂CASPc（chloroaluminum su lfonated phthalocyanine,CASPc）对B16F10黑色素瘤动物模型的光动力治疗（PDT）作用 。 方法 38只新英格兰大白兔经免疫抑制后种植B16F10黑色素瘤碎片于脉络膜下腔，B型超声、间接检眼镜随访肿瘤生长至2.0 ~3.8 mm厚时30只兔眼给予PDT治疗。耳静脉注射CASPc 5 mg/kg, 24 h后给予氩-染料激光照射，激光波长675 nm, 输出功率600 nm/cm2，激光照射剂量20~70 J/cm2；8只对照组兔眼只给予激光或光敏物质。治疗后观察6~8周。 结果 38只兔眼建模成功。30只PDT治疗兔眼中21只眼肿瘤得到控制，9只眼未能控制；对照组兔眼肿瘤治疗后2周充满整个玻璃体。 结论 第二代光敏剂CASPc对实验性眼B16F10黑色素瘤具有一定的PDT治疗作用。 （中华眼底病杂志，2004，20：67-132）     

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.     

早期行保存板层角巩膜联合干细胞植治疗眼严重碱烧伤的临床观察

目的评价早期行保存板层角巩膜移植联合干细胞移植治疗眼严重碱烧伤的疗效。方法将符合Roper-Hall诊断标准Ⅲ～Ⅳ度的眼部碱烧伤患者共28例32眼在受伤二周内行保存板层角巩膜移植联合干细胞移植术。结果随访3～36个月,术后视力提高者18眼(56.2%,18/32),未提高者10眼,下降者4眼。角膜植片透明14眼,透明率43.7%(14/32)。无一眼发生角膜溃疡穿孔。结论对眼严重碱烧伤的患者行早期清创合并保存板层角巩膜移植联合干细胞移植术,可有效减少角膜溃疡穿孔、角膜血管化,提高视力。     

组织工程上皮移植对碱烧伤角膜新生血管的影响

目的：评价组织工程上皮移植在碱烧伤角膜缘干细胞缺乏症中对角膜新生血管的抑制作用。

方法：回顾性非随机的病例研究。2006/2011年我院收治的19例(23眼)完全性角膜缘干细胞缺乏症的碱烧伤患者, 10例13眼行组织工程上皮移植,9例10眼行羊膜移植。所有患者在手术前后均用裂隙灯观察角膜新生血管情况,在术后第21,60d对角膜新生血管进行评分比较。

结果：术后第21d和术后第60d组织工程上皮移植组和羊膜移植组角膜新生血管均较术前明显减少( P<0.05),在术后两个评价时间点,组织工程上皮移植组平均角膜新生血管分数明显低于羊膜移植组。

结论：对碱烧伤所致角膜缘干细胞缺乏的患者,组织工程上皮移植抑制角膜新生血管的作用明显好于羊膜移植。     

中性粒细胞在不同品系正常小鼠角膜的分布及其与角膜创伤修复的关系

背景 以往人们通常认为角膜无血管、无髓系来源的免疫细胞存在.C57BL/6J小鼠、BALB/c小鼠和裸鼠是眼科免疫学基础研究的常用模型,这些小鼠的角膜是否存在天然免疫的关键细胞——中性粒细胞是值得关注的问题. 目的 研究实验室常用的C57BL/6J鼠、BALB/c鼠和裸鼠正常角膜的中性粒细胞分布特征及其与角膜创伤修复的关系,为相关研究提供依据. 方法 选取角膜正常、10 ～ 12周龄的SPF级雄性C57BL/6J鼠、BALB/c鼠和BALB/c背景的裸鼠各16只,取3种小鼠各8只制备成中央区相连的4瓣角膜铺片,并以角膜周边血管缘为界向内以3个同心圆分区.分别用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体对角膜中性粒细胞和血管进行免疫荧光染色,用免疫荧光显微镜和AR软件测量角膜血管面积并计数中性粒细胞.选取3种小鼠各8只制备角膜创伤模型,用无菌手术刀片以角膜中央为中心做十字划痕,深度至角膜前弹力层.创伤后即刻（0 h）、12h、24 h用2 g/L荧光素钠点眼,荧光显微镜下以AR软件计算不同时间点的创伤面积.于划痕后24 h取小鼠角膜制备铺片,用Gr-1-FITC抗体和CD31/PECAM-1-PE抗体行荧光染色,比较3种创伤小鼠角膜的血管分布、中性粒细胞的数量和分布情况.实验动物的饲养与使用均遵循美国视觉与眼科研究协会制定的科研动物使用规范. 结果 正常C57BL/6J小鼠、BALB/c小鼠和裸鼠角膜中性粒细胞总数分别为（1 733±237）、（353±96）和（1 601 ±223）个/角膜,BALB/c小鼠角膜中性粒细胞数明显少于C57BL/6J小鼠和裸鼠,差异均有统计学意义（P＜0.01）.3种正常小鼠角膜缘均可见血管分布,BALB/c小鼠角膜缘血管面积明显小于C57BL/6J小鼠和裸鼠,C57BL/6J小鼠角膜缘血管面积明显大于裸鼠,差异均有统计学意义（P＜0.01）.3种正常小鼠中性粒细胞的数量与血管面积均呈明显正相关（C57BL/6J：r=0.936,P=0.001;BALB/c：r =0.939,P=0.001;     

Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases.