Metabolomics Analyses of Mouse Retinas in Oxygen-Induced Retinopathy

PurposeRetinal neovascularization is a severe pathological process leading to irreversible blindness. This study aims to identify the altered metabolites and their related pathways that are involved in retinal neovascularization.MethodsTo reveal the global metabolomic profile change in the retinal neovascularization process, an untargeted metabolomics analysis of oxygen-induced retinopathy (OIR) mice retinas was carried out first, followed by the validation of amino acids and their derivatives through a targeted metabolomics analysis. The involved pathways were predicted by bioinformatic analysis.ResultsBy untargeted metabolomics, a total of 58 and 49 metabolites altered significantly in OIR retinas under cationic and anionic modes, respectively. By bioinformatics analysis, “ABC transporters,” “central carbon metabolism in cancer.” and “alanine, aspartate, and glutamate metabolism” were the most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with the changed metabolites. By targeted metabolomics, no significant change was found in the assessed amino acids and their derivatives at postnatal day (P) 12, whereas significantly altered amino acids and their derivatives were recognized at P13, P17, and P42 in OIR retinas.ConclusionsThe metabolomic profile was significantly altered in the neovascularized retinas. In particular, numerous amino acids and their derivatives were significantly changed in OIR retinas. These altered metabolites, together with their associated pathways, might be involved in the pathogenesis of retinal neovascular diseases.     

Nattokinase Attenuates Retinal Neovascularization Via Modulation of Nrf2/HO-1 and Glial Activation

PurposeNattokinase (NK), an active ingredient extracted from traditional food Natto, has been studied for prevention and treatment of cardiovascular diseases due to various vasoprotective effects, including fibrinolytic, antihypertensive, anti-atherosclerotic, antiplatelet, and anti-inflammatory activities. Here, we reported an antineovascular effect of NK against experimental retinal neovascularization.MethodsThe inhibitory effect of NK against retinal neovascularization was evaluated using an oxygen-induced retinopathy murine model. Expressions of Nrf2/HO-1 signaling and glial activation in the NK-treated retinae were measured. We also investigated cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) after NK administration.ResultsNK treatment significantly attenuated retinal neovascularization in the OIR retinae. Consistently, NK suppressed VEGF-induced cell proliferation and migration in a concentration-dependent manner in cultured vascular endothelial cells. NK ameliorated ischemic retinopathy partially via activating Nrf2/HO-1. In addition, NK orchestrated reactive gliosis and promoted microglial activation toward a reparative phenotype in ischemic retina. Treatment of NK exhibited no cell toxicity or anti-angiogenic effects in the normal retina.ConclusionsOur results revealed the anti-angiogenic effect of NK against retinal neovascularization via modulating Nrf2/HO-1, glial activation and neuroinflammation, suggesting a promising alternative treatment strategy for retinal neovascularization.     

阻断促红细胞生成素抑制小鼠视网膜新生血管生成

目的：建立C57 BL/6小鼠氧致血管增生性视网膜病变模型( oxygen induced retinopathy, OIR),探讨阻断促红细胞生成素( Erythropoietin, EPO )对视网膜新生血管的抑制作用。
　　方法：取鼠龄7 d ( P7)的健康C57 BL/6小鼠置于75%±2%氧气浓度的密闭氧舱中5d,鼠龄12d (P12)时返回正常空气环境以建立氧诱导鼠视网膜新生血管模型;新生小鼠于P12~P16隔日给予玻璃体腔内注射0.5μL含有25ng(A组),50ng(B组),250ng(C组)可溶性EPO受体的PBS液以及不含EPO受体的PBS液( D组)。于P17时分批处死各组小鼠,小鼠眼球以4%多聚甲醛固定,制成病理切片,进行视网膜组织形态病理学观察计数突破内界膜新生血管细胞核数,了解视网膜新生血管增生程度。
　　结果：计数病理切片突破内界膜新生血管细胞核数目,各组玻璃体内EPO受体注射组较PBS注射组新生血管细胞核数明显减少,差异有统计学意义(P<0.01)。并且各不同浓度EPO受体组间新生血管细胞核计数差异亦有统计学意义(P<0.01),随着EPO受体浓度增高,突破内界膜新生血管内皮细胞数减少。
　　结论：可溶性EPO受体玻璃体腔内注射能够阻断EPO的促新生血管生成作用,有望成为治疗眼部新生血管性疾病的新方法。     

P16在氧诱导视网膜病变大鼠视网膜中的表达及作用机制

目的:研究氧诱导视网膜病变(oxygen-induced retinopathy,OIR)中p16表达的变化对新生血管增殖的影响.方法:鼠龄7d的SD大鼠60只随机分为正常组、模型组、干预组和NS对照组共4组.正常组置于正常空气中饲养;模型组置于75mL/L高氧环境5d建立氧诱导视网膜病变模型;干预组给予抗p16甲基化药物5-aza-CdR(0.25mg/kg)腹腔注射;NS对照组腹腔注射同体积的生理盐水.各组分别取双侧眼球,左眼做HE染色观察视网膜新生血管,免疫组织化学和免疫荧光观察p16蛋白的表达.右眼视网膜行real time-PCR分析p16 mRNA的表达.结果:正常组幼鼠的视网膜未发现突破视网膜内界膜的新生血管管腔.模型组和NS对照组视网膜组织明显增厚,可见大量新生血管管腔.干预组视网膜偶见新生血管管腔.免疫组织化学和免疫荧光显示,模型组视网膜p16呈低表达,阳性细胞数为19.52±2.67个;而干预组阳性细胞数为36.38±3.16个,差异有统计学意义(P＜0.001).real time-PCR显示,模型组p16 mRNA的表达降低(2-△△ct=0.14±0.01),p16 mRNA在干预组大鼠的视网膜表达明显高于NS对照组大鼠视网膜,两组相比差异有统计学意义(2-△△ct=0.68±0.08,P＜0.001).结论:P16的异常表达可能与视网膜新生血管增殖密切相关,抑制p16甲基化可减少视网膜新生血管的增殖.     

巨噬细胞在小鼠氧诱导视网膜新生血管形成中的作用

目的　探讨巨噬细胞对小鼠氧诱导视网膜病变（ｏｘｙｇｅｎ-ｉｎｄｕｃｅｄｒｅｔｉｎｏｐａｔｈｙ,ＯＩＲ）形成的影响。方法　将新生Ｃ５７ＢＬ／６Ｊ小鼠随机分为３组,分别为常氧对照组、ＯＩＲ模型组以及清除巨噬细胞的ＯＩＲ组。将后两组小鼠于生后第７天（Ｐ７）至Ｐ１２置于体积分数（７５±２）％高氧氧箱中以诱导ＯＩＲ。清除巨噬细胞的ＯＩＲ组小鼠于Ｐ９、Ｐ１１、Ｐ１３和Ｐ１５接受腹腔注射氯膦酸二钠脂质体４次以清除全身单核-巨噬细胞,ＯＩＲ模型组小鼠则于上述４个时间点接受腹腔注射ＰＢＳ脂质体。三组小鼠均于Ｐ１７时取右眼行视网膜铺片和Ｌｅｃｔｉｎ染色,观察视网膜出血及血管生长情况;取左眼行视网膜切片和ＨＥ染色,观察视网膜组织病理学变化。结果　与常氧对照组相比,ＯＩＲ模型组小鼠视网膜存在出血现象,清除巨噬细胞的ＯＩＲ组出血有所减轻。ＯＩＲ模型组小鼠视网膜血管形态呈异常增殖的团簇状,走行迂曲,而清除巨噬细胞的ＯＩＲ组小鼠视网膜新生血管异常形态减轻。与ＯＩＲ模型组相比,清除巨噬细胞的ＯＩＲ小鼠视网膜无血管区及新生血管区面积均显著减小［（１７．１９±０．５８）％、（１０．３８±０．５３）％,ｔａ＝８．６８０,Ｐ＜０．０１;（４．６０±０．１５）％、（２．５１±０．１３）％,ｔｎ＝１０．８３,Ｐ＜０．０１］,突破内界膜新生血管内皮细胞核数目明显减少（１８．５０±０．８５、７．１７±０．４８;ｔ＝１１．６６,Ｐ＜０．０１）。结论　清除巨噬细胞可减轻小鼠ＯＩＲ严重程度,提示巨噬细胞对视网膜新生血管形成具有促进作用。     

The progress of prophylactic treatment in retinopathy of prematurity

Retinopathy of prematurity (ROP) is a retinal vascular disorder frequently found in premature infants. Different therapeutic strategies have been developed to treat ROP. However, there are still many children with ROP suffering by severe limitations in vision or even blindness. Recently, ROP has been suggested to be caused by abnormal development of the retinal vasculature, but not simply resulted by retinal neovascularization which takes about 4 to 6wk after birth in premature infants. Thus, instead of focusing on how to reduce retinal neovascularization, understanding the pathological changes and mechanisms that occur prior to retinal neovascularization is meaningful, which may lead to identify novel target(s) for the development of novel strategy to promote the healthy growth of retinal blood vessels rather than passively waiting for the appearance of retinal neovascularization and removing it by force. In this review, we discussed recent studies about, 1) the pathogenesis prior to retinal neovascularization in oxygen-induced retinopathy (OIR; a ROP in animal model) and in premature infants with ROP; 2) the preclinical and clinical research on preventive treatment of early OIR and ROP. We will not only highlight the importance of the mechanisms and signalling pathways in regulating early stage of ROP but also will provide guidance for actively exploring novel mechanisms and discovering novel treatments for early phase OIR and ROP prior to retinal neovascularization in the future.     

小鼠氧诱导视网膜新生血管形成中促红细胞生成素的变化

目的:观察C57BL/6小鼠氧诱导视网膜新生血管病变模型(oxygen-induced retinopathy,OIR)眼内促红细胞生成素(erythropoietin,EPO)含量的变化。方法:新生C57BL/6小鼠20只,随机分为实验组(n=10)和对照组(n=10)。实验组小鼠于出生后7d(P7)与母鼠共置于密闭的氧箱,含氧75±5mL/L,共培养5d(P5)后回到正常氧环境,含氧20±1mL/L,建立OIR模型。对照组小鼠不进入氧箱,与母鼠一同饲养于正常氧环境。于P17处死各组小鼠,摘取右眼眼球制作组织切片,HE染色后行病理组织学检查,计数突破内界膜视网膜新生血管(retinal neovascularization,RNV)内皮细胞核数。采用放射免疫法测定实验组和对照组小鼠左眼视网膜组织EPO含量。结果:实验组小鼠视网膜突破内界膜RNV内皮细胞核计数为80.0±6.2个,而对照组仅为1.0±0.9个,两者差异有统计学意义(P<0.01)。实验组视网膜EPO含量为80.8±20.7U/L,对照组为14.4±6.8U/L,两者差异有统计学意义(P<0.01),且视网膜组织EPO表达与RNV增生程度呈明显正相关(r=0.58,P<0.01)。结论:小鼠视网膜新生血管形成与眼球局部EPO上调有关。     

精氨酸-谷氨酰胺在幼鼠早产儿视网膜病变模型中的应用(英文)

目的:观察精氨酸-谷氨酰胺(Arg-Gln)对早产儿视网膜病变动物模型视网膜新生血管的抑制作用。方法:48只7日龄的C57BL/6J新生鼠暴露在750mL/L高氧环境中5d,然后回到正常空气中建立早产儿视网膜病变的动物模型。在鼠龄12d时实验组(36只)新生鼠每天两次腹腔注射Arg-Gln(剂量分别为1.0,3.0,5.0g/kg,每组12只),连续注射5d;对照组(12只)每天两次腹腔注射PBS,连续5d。所有小鼠均于17d处死,视网膜铺片,ADP酶染色观察视网膜血管情况。HE染色,在光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞细胞核数目。Real-time RT-PCR方法测量每组视网膜VEGF mRNA水平。结果:与对照组相比,实验组以剂量依赖方式无灌注区面积和新生血管团逐渐减少;实验组中最大剂量组[5.0g/(kg·d)]突破内界膜的内皮细胞细胞核数目比对照组大约减少75%(P<0.01);实验组视网膜VEGF mRNA水平与对照组相比明显下降。结论:Arg-Gln能够有效抑制早产儿视网膜病变动物模型视网膜新生血管的生成,可能为临床提供一种预防和治疗早产儿视网膜病变安全有效的新方法。     

Effect of the dipeptide Arg-Gln on retinopathy of prematurity in mice

AIM: To investigate the effect of the dipeptide Arg-Gln on retinal neovascularization of retinopathy of prematurity (ROP) in the oxygen-induced retinopathy (OIR) animal model. METHODS: Forty-eight 7-day-old C57BL/6J mice were exposed to 750mL/L oxygen for 5 days and then to normal situation to produce the murine model of oxygen-induced retinopathy (OIR). All mice received twice daily intra- peritoneal injections of PBS or the dipeptide Arg-Gln (1.0, 3.0, 5.0g/kg per day), starting on postnatal day 12 and continuing till postnatal day 17. Experimental groups (36 mice, 12 in each group) received Arg-Gln, while the control group (12 mice) received PBS. All mice were executed at postnatal day 17. The changes of retinal vessels of mice were observed by ADPase histochemical technique and HE staining was used to count preretinal neovascular nuclei. RNA was isolated from retinas of 28 mice (7 in each group) selected at random and VEGF mRNA level of each group was measured by real-time RT-PCR. RESULTS: Neovascularization reduced in retinas of the dipeptide Arg-Gln treated group in a dose-dependent manner. Compared with control group, experimental group had diminished non-perfusion area and neovascular tufts in retinal flatmount. The number of the endotheliocyte nuclei of new vessels extending from retina to vitreous was significantly less in the eyes of the experimental group than in control group. Arg-Gln at 5g/kg per day reduced preretinal neovascularization by about 75% (P <0.01). There was a significant reduction in VEGF mRNA at the 17^th day in Arg-Gln treated group compared with control group(P ＜0.01). CONCLUSION: Arg-Gln dramatically inhibits retinal angiogenesis in OIR and this effect is associated with a reduction in retinal VEGF mRNA level. It appears to be a safe way to prevent and treat some neovascular retinal diseases including retinopathy of prematurity.     

Matrix metalloproteinase-9 and vascular endothelial growth factor expression change in experimental retinal neovascularization

AIM: To investigate the signal transduction mechanism of matrix metalloproteinase-9 (MMP-9) mediated- vascular endothelial growth factor (VEGF) expression and retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) model. METHODS: C57BL/6J mice were divided into four groups: control group, OIR group, OIR control group (phosphate-buffered saline by intravitreal injection) and treated group [tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by intravitreal injection]. OIR model was established in C57BL/6J mice exposed to 75%±2% oxygen for 5d. mRNA level and protein expression of MMP-9, TIMP-1 and VEGF were measured by real-time polymerase chain reaction and Western blotting, and located by immunohistochemistry. RESULTS: Levels of MMP-9 and VEGF in retina were significantly increased in animals with OIR and OIR control group. Levels of TIMP-1 in retina was significantly reduced in animals with OIR and OIR control group. Furthermore, a significant correlation was found between MMP-9 and VEGF. Intravitreal injection of TIMP-1 significantly reduced MMP-9 and VEGF expression of the OIR mouse model (all P<0.05). CONCLUSION: These results demonstrate that MMP-9-mediated up-regulation of VEGF promotes RNV in retinopathy of prematurity (ROP). TIMP-1 may be a potential target for the prevention and treatment of ROP.     

Efficacy of intravitreal captopril on oxygen-induced retinopathy in mice

AIM: To study the inhibitory effect of intravitreal captopril on oxygen-induced retinopathy (OIR) in mice. METHODS: Eighty postnatal day (P)7 C57BL/6J mice were randomly divided into treated group and control group with forty mice in each group. The mice were exposed to 75% ± 2% oxygen for 5 days (P7-P11) and then returned to room air for 5 days (P12-P17) to induce retinal neovascularization (RNV). Beginning on P12, the mice in treated group received daily intravitreal injections of captopril (3.0mL/kg), while those in control group received daily intravitreal injections of phosphate-buffered saline (PBS) (3.0mL/kg) through P17. After anesthetized at P17, one eye was chosen randomly as experimental eye and were enucleated. RNV was examined by Adenosine diphosphate-ase (ADPase) stained retina flat-mounts and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to inner limiting membrane (ILM). The expressions of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were measured by immunohistochemical method. RESULTS: Comparing with control group, more regular distributions, better branch and reduced density of RNV were observed in eyes of treated group. The number of neovascular cell nuclei was less in treated group than that in control group (t=6.135, P<0.01). Stain of MMP-2 and VEGF was weaker in treated group than that in control group. CONCLUSION: The results indicate that captopril can significantly inhibit RNV in OIR mice.     

氧诱导小鼠视网膜新生血管发生中乙酰肝素酶及串珠素的表达

目的研究小鼠视网膜新生血管发生过程中乙酰肝素酶（HPA）及其作用底物串珠素在视网膜中的表达。方法将65只新生幼鼠于生后7d在体积分数（75±2）％高氧环境中饲养5d后,再置于相对低氧环境中诱导产生视网膜新生血管为高氧诱导组。另外65只新生幼鼠在正常环境中饲养作为正常对照组。分别在小鼠生后第12、13、17、21、30天通过荧光素眼底血管造影（FFA）和视网膜病理切片观察视网膜新生血管的形态变化。通过逆转录聚合酶链反应（RT—PCR）分别检测不同时间点视网膜组织中HPA和串珠素在mRNA水平的变化,Westernblot检测不同时间点视网膜组织中HPA在蛋白水平的表达变化。结果高氧诱导组与正常对照组的HPAmRNA表达水平差异有统计学意义（Fgroup=16．303,P=0．000）,各时间点的HPAmRNA表达水平差异有统计学意义（Ftime=18．614,P=0．000）,生后第12、13、17、21天相应时间点2组小鼠视网膜HPAmRNA的表达水平差异均有统计学意义（P=0．001,P=0．000,P：0．000,P=0．001）。高氧诱导组与正常对照组HPA蛋白表达水平差异有统计学意义（Fgroup=458．134,P=0．000）,各时间点的HPA蛋白表达水平差异有统计学意义（F time=78．466,P=0．000）。高氧诱导组与正常对照组的串珠素mRNA表达水平差异有统计学意义（Fgroup=7．351,P=0．013）,各时间点的串珠素mRNA表达水平差异有统计学意义（Ftime=9．098,P=0．000）。生后第13、17、21天的高氧诱导组小鼠视网膜串珠素mRNA的表达水平与正常对照组相应时间点,差异均有统计学意义（P=0．048,P=0．000,P=0．003）。伴随着视网膜新生血管的增多和消退,HPA在基因和蛋白水平及串珠素在基因水平的表达均呈先升高后降低的趋势。结论HPA及其作用底物可能是促进视网膜新生血管生长的重要因素。     

Arresten在氧诱导小鼠视网膜新生血管形成中的抑制作用及机制

目的　探讨Arresten在氧诱导小鼠视网膜新生血管形成中的抑制作用及机制。方法　选取48只出生后7 d健康清洁级C57BL/6J幼鼠,随机分为氧诱导视网膜病变（oxygen induced retinopathy,OIR）组、OIR+ Arresten组和常氧组,每组16只。OIR+Arresten组及OIR组幼鼠在体积分数为（75±2）％的高氧环境下饲养5 d,然后转移至正常氧气环境中饲养5 d,其中OIR+Arresten组幼鼠于高氧饲养刚结束时,给予双眼玻璃体内注射Arresten蛋白。常氧组幼鼠则在常规氧气环境中连续饲养10 d。在鼠龄17 d时,各组取6只幼鼠经股静脉注射异硫氰酸葡聚糖溶液处死并摘出眼球,视网膜铺片后观察视网膜的血管形态、计算无灌注区面积。同时对小鼠眼球视网膜行石蜡切片和HE染色,计算侵入玻璃体内血管内皮细胞核的数量。同时,通过细胞培养实验,利用MTT法检测Arresten蛋白对人血管内皮细胞增殖的抑制作用。结果　视网膜铺片结果显示,常氧组、OIR组、OIR+Arresten组视网膜无灌注区相对面积分别为（2.35±1.62）％、（57.28±9.36）％和（20.38±8.69）％,三组间总体比较,差异有统计学意义（F=18.732,P<0.05）,且OIR组无灌注区相对面积显著高于OIR+Arresten组,差异有统计学意义（P<0.05）。常氧组小鼠视网膜的内界膜结构仍保持平滑完整,未发现有新生血管侵入玻璃体内。OIR组和OIR+Arresten组每张切片上血管内皮细胞核的数量分别为 （15.18±4.83）个、（7.33±3.88）个,其中OIR+Arresten组侵入玻璃体内新生血管内皮细胞核的数量显著低于OIR组,差异有统计学意义（P<0.05）。Arresten蛋白对人脐静脉内皮细胞增殖的抑制率随着其浓度的升高而增加,但当Arresten蛋白浓度达到1000 μg·L^-1时,人脐静脉内皮细胞的增殖抑制率达到顶峰,为（58.00±0.65）％。结论　Arresten蛋白能够抑制氧诱导的小鼠视网膜新生血管形成,通过抑制血管内皮细胞增殖来抑制新生血管形成。     

Caspase-1对氧诱导视网膜病变中小胶质细胞参与视网膜新生血管生成的促进作用及其机制

目的：探讨Caspase-1对小鼠氧诱导视网膜病变(oxygen-induced retinopathy,OIR)中小胶质细胞参与视网膜新生血管生成的作用及其机制。

方法：随机将12只7日龄(P7)C57BL/6J小鼠分为正常组、OIR组和OIR+VX-765组,正常组在正常氧环境中饲养,其余两组构建OIR模型; P12～P16,OIR+VX-765组和OIR组分别每天腹腔注射Caspase-1抑制剂VX-765(4mg/kg)和等量0.4%聚乙二醇(VX-765溶剂); 于P17制作视网膜铺片行Lectin染色,比较三组间视网膜无血管区和新生血管区面积的大小; 采用免疫荧光染色法观察视网膜组织中Caspase-1的表达和活化小胶质细胞的分布。培养小胶质细胞BV-2细胞分为对照组、缺氧组及抑制剂组,抑制剂组和缺氧组分别经VX-765和0.4%聚乙二醇预处理3h后,缺氧条件下培养24h; 对照组常规培养相同时间。通过Western blot检测Caspase-1、p20(Caspase-1活化形式)、IL-1β和VEGF的蛋白表达变化; 用各组BV-2细胞培养上清液作为条件培养基,刺激培养血管内皮细胞RF/6A,进行管腔形成和细胞迁移实验,并比较各组间的差异。

结果：P17正常组小鼠视网膜血管化完全,未见明显无血管区及视网膜新生血管; OIR组视网膜无血管区和新生血管面积百分比分别为16.58%±1.14%、4.00%±0.41%; OIR+VX-765组两者明显减少,分别为12.23%±1.02%和2.16%±0.52%(P<0.01)。免疫荧光染色结果显示,Caspase-1在正常小鼠视网膜组织中表达较弱,在OIR小鼠中主要在神经节细胞层和内丛状层有明显的阳性表达,并与活化的小胶质细胞有明确的共定位。Western blot检测结果显示,缺氧处理的培养小胶质细胞BV-2中Caspase-1、p20、IL-1β和VEGF蛋白表达明显提高,而Caspase-1抑制剂则可明显下调p20、IL-1β和VEGF的蛋白表达水平(P<0.05)。管腔形成和细胞迁移实验结果显示,RF/6A细胞经缺氧组BV-2培养上清液处理后,管腔形成长度和细胞迁移数目分别为271±12和347±34个,而加入抑制剂后,二者明显减少,分别为171±22和212±27个(P<0.05)。

结论：在小鼠OIR中,Caspase-1能够调节小胶质细胞促进视网膜新生血管的生成,其作用机制可能与Caspase-1活化小胶质细胞中其下游炎性效应分子IL-1β,并释放VEGF相关。     

两种检测小鼠氧诱导视网膜新生血管方法的比较

目的比较异凝集素视网膜铺片染色和硫氰酸葡聚糖荧光素心脏灌注视网膜铺片两种方法检测氧诱导视网膜病变(OIR)新生血管的优缺点,探讨视网膜新生血管染色方法的合理选择。设计实验研究。研究对象C57BL/6J乳鼠OIR模型8只,正常对照8只。方法 OIR组和正常对照组各4只鼠视网膜铺片异凝集素染色,各4只鼠硫氰酸葡聚糖荧光素心脏灌注视网膜铺片。3位观察者盲法利用图像分析软件计算视网膜铺片上视网膜血管无灌注区和新生血管面积。主要指标不同观察者计算的视网膜新生血管及无灌注区面积的一致性(α值)。结果异凝集素染色视网膜铺片上,不同观察者计算的视网膜新生血管及无灌注区面积α值分别为0.858、0.959;硫氰酸葡聚糖荧光素心脏灌注视网膜铺片上,不同观察者计算的视网膜新生血管及无灌注区面积α值分别为0.626、0.972。结论利用OIR模型研究视网膜新生血管时,异凝集素染色视网膜铺片和硫氰酸葡聚糖荧光素心脏灌注视网膜铺片均能清晰显示新生血管和无灌注区,前者在量化视网膜新生血管和无灌注区面积时均较可靠,而后者只适合于评价视网膜新生血管的灌注情况。     

New quantitative analysis, using high-resolution images, of oxygen-induced retinal neovascularization in mice

Oxygen-induced retinopathy (OIR) has been widely studied as an animal model of retinal neovascularization diseases. Evaluation using this model is mainly performed by counting cell nuclei above the internal limiting membrane in serial cross-sections or by scoring in flat-mounted retinas. Quantitative evaluation is important for accurate elucidation of pathological conditions and the drug evaluations. We therefore attempted quantification using new imaging software and high-resolution images taken with a high-resolution CCD camera. Neonatal mice were exposed to 75% oxygen from postnatal day 7 (P7) to P12, then returned to room air until P17. At each evaluation time, mice were perfused with fluorescein-dextran, and flat-mounted retinas were prepared. Total images of the retinal vasculature were collected and analyzed using the imaging software. P17 normal retinas showed increases in computerized total tube area, total tube length, number of segments, and number of branch points (versus P7 normal retinas). These increases coincided with the development of the retinal vasculature between P7 and P17. P17 OIR retinas similarly showed increases in those parameters, and the number of nodes (thick regions exceeding the maximum width of the vessel) and the node area (abnormality induced by OIR) were markedly increased (versus P17 normal retinas). Accordingly, this approach is considered most suitable for evaluating the number of nodes and node area in this model. Quantification using the present imaging software should be useful for evaluating physiological and pathological neovascularizations in this OIR model.