Reduced severity of oxygen-induced retinopathy in the newborn rat after topical administration of timolol maleate

The effects of timolol maleate on intraocular pressure (IOP) and the severity of retinopathy induced by exposure to 80% oxygen were studied in newborn Wistar rats. One drop of timolol maleate (0.25%) instilled in each eye twice a day for the first ten days of life substantially reduced intraocular pressure without significantly modifying arterial pressure. Forty percent of the ratlings treated in this way failed to develop oxygen-induced retinopathy (OIR) after exposure to 80% oxygen for the first five days of life; in the other 60% OIR was less severe than that seen in an identically oxygenated group that did not receive timolol. The authors hypothesize that the pharmacologically induced reduction in IOP may have attenuated the effects of the high concentrations of oxygen on the immature retinal vessels by improving the ocular perfusion pressure. The possibility that timolol maleate also exerts a direct action on the caliber of these vessels cannot be excluded.Abbreviations BP blood pressure - OIR oxygen-induced retinopathy     

单核细胞趋化蛋白-1在氧诱导视网膜病变新生小鼠模型中的表达

背景 单核细胞趋化蛋白-1( MCP-1)是趋化因子家族中的主要成员之一,在肿瘤、炎症和糖尿病视网膜病变(DR)等新生血管性疾病中起着重要的作用,但关于MCP-1在氧诱导视网膜病变(OIR)发生过程中的表达及其作用的研究鲜有报道. 目的 观察OIR小鼠视网膜中MCP-1的表达,探讨MCP-1在视网膜血管发育和视网膜新生血管形成过程中的作用. 方法 SPF级C57BL/6J小鼠120只,按随机数字表法随机分为OIR组和正常对照组,每组60只.OIR组将出生后7d的新生小鼠在体积分数75％氧环境下饲养5d,然后返回正常大气环境中;正常对照组小鼠始终置于正常大气环境中饲养.OIR组和正常对照组分别在出生后5、7、12、14、17、21 d随机抽取10只小鼠,摘除右眼球,采用免疫组织化学法检测MCP-1蛋白在小鼠视网膜中的表达情况,采用逆转录-聚合酶链反应(RT-PCR)法检测MCP-1 mRNA在小鼠视网膜中的表达. 结果 正常对照组小鼠在出生后的第5天即有MCP-1在视网膜的内核层和节细胞层表达,12d时表达MCP-1的阳性细胞数达到高峰,其他日龄时呈基线表达.OIR组12d时表达MCP-1的阳性细胞数轻度增多,14d时阳性细胞数显著增多,之后迅速下降至正常水平.正常对照组在5d时可检测到MCP-1 mRNA表达,12d时显著上调,之后随小鼠日龄的增加,MCP-1 mRNA表达迅速下降,14、17、21 d表达基本呈基线水平.OIR组12 d时,MCP-1 mRNA较正常对照组下降,在新生血管形成关键期,即14 d时表达显著上调,之后迅速下降至正常水平.OIR组和正常对照组间比较采用完全随机分组的两因素方差分析,F分组=5.230,P=0.028;F日龄=6.898,P=0.001.结论 小鼠视网膜发育过程始终伴随着MCP-1的表达,MCP-1的表达上调可能与小鼠视网膜血管发育和OIR模型中视网膜新生血管的形成密切相关.     

Variable oxygen and retinal VEGF levels: correlation with incidence and severity of pathology in a rat model of oxygen-induced retinopathy

Retinal capillary quiescence is regulated by a delicate balance between proangiogenic and anti-angiogenic factors. Pathological angiogenesis is the result of a shift in this balance towards proangiogenic influences. Pathological angiogenesis is produced in a rat model of oxygen-induced retinopathy (OIR) by exposing newborn rat pups to alternating periods of hyperoxia and hypoxia. Based upon previous work, two similar exposure paradigms were investigated and compared, exposure of rat pups to alternating periods of 45 and 12.5% oxygen, and to alternating periods of 40 and 15% oxygen. The resulting retinal pathology was assessed by measurement of retinal clock hours with pathological blood vessel growth and the percentage of the retina that is avascular. The 45 and 12.5% exposure produced significantly greater incidence and severity of pathology than the 40 and 15% protocol. To explain the difference in pathology between these two very similar exposure protocols, retinal levels of proangiogenic vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) and anti-angiogenic pigment epithelium-derived factor (PEDF) were measured by ELISA and western blot analysis at 0, 2, and 6 days post-exposure. In whole retinal lysates, there were no significant differences in VEGFR2 and PEDF levels. However, VEGF levels were approximately 48 and 78% higher on post-oxygen exposure day 0 and 2, respectively, in the group treated with alternating periods of 45 and 12.5% oxygen compared to the group treated with alternating periods of 40 and 15% oxygen. There was no significant difference in VEGF levels between these two groups on day 6 post-exposure. Therefore, the difference in pathology observed between these two experimental paradigms is associated with differences in whole retinal VEGF levels, but not changes in whole retinal VEGFR2 or PEDF levels. The results of this study suggest the existence of a threshold in the rat model of OIR, such that a small change in blood oxygen profile triggers a disproportionate increase in subsequent neovascularization, which is accompanied by more dramatic changes of retinal VEGF level than VEGFR2 or PEDF level. If a similar threshold exists for humans, it could explain why some oxygen-treated premature infants develop retinopathy and others do not, despite similar gestational ages, birth weights and clinical courses.     

夜间光照对氧诱导的视网膜病变小鼠视网膜新生血管形成的影响

背景 氧诱导的视网膜新生血管形成是多种视网膜血管性疾病的病理学基础,预防视网膜新生血管的形成可缓解视网膜病变对视网膜的损害程度.研究表明夜间睡眠时给予光照可能对早期糖尿病视网膜病变(DR)患者有利,但其对早产儿视网膜病变(ROP)患者视网膜新生血管有无影响报道较少.目的 观察夜间光照对氧诱导视网膜病变(OIR)小鼠视网膜新生血管形成的影响.方法 将64只SPF级新生C57BL/6J小鼠按随机数字表法随机分为正常对照组、单纯夜间光照组、OIR模型组、OIR联合夜间光照组,每组16只小鼠.正常对照组和单纯夜间光照组小鼠生长于正常空气(氧体积分数21％);OIR模型组和OIR模型联合夜间光照组小鼠于出生后第7天置于高氧环境(75％±2％)生长,出生第12天调整氧体积分数为正常;OIR联合夜间光照组和单纯夜间光照组于出生后第12～17天给予夜间光照,光照度为100 Ix.各组小鼠均于出生后第17天摘除眼球,采用ADP酶法制备视网膜铺片,了解视网膜血管的改变情况;视网膜组织切片行苏木精-伊红染色并计数突破视网膜内界膜的新生血管内皮细胞核数;免疫组织化学法观察各组小鼠视网膜中血管内皮生长因子(VEGF)的表达,实时荧光定量聚合酶链反应(PCR)法检测各组视网膜中VEGFmRNA的表达.实验动物的使用和喂养遵循ARVO声明.结果 正常对照组和单纯夜间光照组小鼠视网膜血管形态均无明显差异.OIR模型组视网膜铺片显示视网膜中央部大片无血管区,大量结构异常的新生血管形成.与OIR模型组相比,OIR模型联合夜间光照组视网膜中央无血管区面积以及新生血管分布密度减少.在实验后第17天时,正常对照组和单纯夜间光照组视网膜新生血管内皮细胞核数分别为(0.97±0.83)个和(1.00±0.72)个,OIR模型组为(38.57±5.01)个,而OIR联合夜间光照组小鼠视网膜新生血管内皮细胞核数为(16.92±3.39)个,总体差异有统计学意义(F=78.767,P=0.000),OIR联合夜间光照组视网膜新生血管内皮细胞核数明显少于OIR模型组,差异有统计学意义(t=20.446,P＜0.01).免疫组织化学法检测显示OIR模型联合夜间光照组中VEGF蛋白表达明显少于OIR模型组.正常对照组、单纯夜间光照组、OIR模型组和OIR联合夜间光照组小鼠视网膜VEGF mRNA相对表达量分别为1.00±0.00、0.94±0.07、2.08±0.50和1.43±0.21,各组间的总体差异有统计学意义(F=11.268,P=0.003),OIR模型联合夜间光照组表达较OIR模型组下调,差异有统计学意义(t=20.163,P＜0.05).结论 夜间光照可减少OIR小鼠视网膜新生血管的形成.     

基质细胞衍生因子-1在氧诱导视网膜病变小鼠视网膜中的表达

背景早产儿视网膜病变（ROP）的进展与多种新生血管调控因子有关,而基质细胞衍生因子·1（SDF．1）在氧诱导视网膜病变（OIR）动物模型视网膜中的表达及其作用机制尚不明确。目的对SDF-1在OIR小鼠模型视网膜中的表达进行定位和定量分析。方法应用随机数字表法将动物随机分组。将20只7日龄C57BL／6J幼鼠暴露在体积分数（75±2）％浓度的高氧状态下5d,随后在正常氧环境下5d,作为OIR组;另20只同日龄幼鼠作为正常对照组。通过免疫组织化学染色和实时荧光定量聚合酶链反应（real-timePCR）法观察视网膜的SDF-1的蛋白表达以及SDF-1mRNA的变化。结果OIR组12日龄小鼠的视网膜神经节细胞层可见SDF．1蛋白呈阳性表达;OIR组17习龄小鼠的神经节细胞层、内层视网膜的血管内皮细胞、新生血管内皮细胞可见SDF-1蛋白呈强阳性表达;正常对照组12日龄小鼠SDF-1蛋白和正常对照组17日龄小鼠SDF-1蛋白微弱表达于内层视网膜及视网膜血管附近。OIR组17日龄小鼠的SDF-1mRNA表达水平明显高于OIR组12日龄小鼠（P〈O．01）及正常对照组17日龄小鼠（P〈0．01）;OIR组12日龄小鼠SDF-1mRNA表达水平明显低于正常对照组12日龄小鼠（P〈0．05）。OIR组17日龄小鼠的SDF-1mRNA表达水平明显高于OIR组12日龄小鼠（t=8．072,P〈0．05）和正常对照组17日龄小鼠（t=10．026,P〈O．05）;OIR组12日龄小鼠SDF-1mRNA表达水平明显低于正常对照组12日龄小鼠（t=4．336,P〈0．05）。结论SDF-1在相对低氧状态下的视网膜中表达明显上调,因而可促进OIR的视网膜新生血管形成。     

Protective effects of triamcinolone acetonide upon the upregulation and phosphorylation of GAP 43 in an animal model of retinopathy of prematurity

Acta Ophthalmol. 2010: 88: e217–e221

Abstract.

Purpose: The aim of the current study was to investigate the effects of triamcinolone acetonide (TA) upon the expression and phosphorylation of growth‐associated protein 43 (GAP 43) in the retinas of oxygen‐induced retinopathy (OIR) rats. Methods: Oxygen‐induced retinopathy was induced by exposing Sprague‐Dawley rats to hyperoxia (80% oxygen) from postnatal (P) days 2–14 and then returning the rats to normoxic conditions. Triamcinolone acetonide or a conditioned saline (control) was injected intravitreally into the right or left eye, respectively, of OIR rats at P15. We then assessed the molecular and histological changes in the expression of GAP 43 and phospho‐GAP 43 in OIR and control rat retinas, and also after treatment with TA by RT‐PCR, Western blotting and immunohistochemistry. Results: Growth‐associated protein 43 mRNA levels were found to be increased by 1.6‐fold (p = 0.001, n = 5) in the retinas of P18 OIR rats compared with the control rats. The protein levels of GAP 43 and phospho‐GAP43 were found to be elevated in the retina of P18 OIR rats (2.40‐ and 2.39‐fold greater than each control, p<0.001, n = 5, respectively). Immunoreactivities of GAP 43 and phospho‐GAP 43 were stronger in the inner plexiform layer in OIR rat retinas compared with the control. However, treatment with TA attenuated GAP 43 and phospho‐GAP 43 upregulation in the OIR retinas. Conclusion: Our results indicate that GAP 43 and phospho‐GAP 43 participate in retinal (potentially pathologic) changes following oxygen‐induced damage. Triamcinolone acetonide protects the retinal damage in relatively hypoxic retinas of OIR rats. Therefore, TA treatment does not induce the expression and phosphorylation of GAP 43 in OIR rat retinas.     

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。     

Effects of hyperbaric, normobaric and hypobaric oxygen supplementation on retinal vessels in newborn rats: a preliminary study

An experimental study was conducted on eight litters of newborn rats to evaluate the effects of supplemental oxygen administration on the retinal vasculature. The animals and their mothers were kept inside a pressure chamber and treated for the first 5 days of life. On the sixth day, they were removed and kept for five more days under room air and normobaric conditions. Three litters received continuous flow oxygen at 80% at a compression pressure of +81 kPa, one litter oxygen at 80% at a pressure of -39.5 kPa atms and three other litters received oxygen at 80% under normobaric conditions. The eighth litter was treated with room air oxygen at a compression pressure of +81 kPa. A severe retinopathy with marked retinal neovascularization was seen only in the newborn animals of the litters that received oxygen supplementation under normobaric or hypobaric conditions. Retinal vessels showed no pathological changes in the litters treated with hyperbaric normoxia or hyperoxia. It is possible to hypothesize that the prolonged period of oxygen supplementation failed to produce harmful effects on the retinal vasculature because the moderate hyperbarism caused mild retinal and choroidal vasoconstriction thus preventing excessive oxygen transport to the inner retina from the choroid during hyperoxia without inducing structural damage to the retinal tissue.     

玻璃体腔注射VAS2870对小鼠氧诱导视网膜病变的保护作用

目的：观察玻璃体腔注射VAS2870对C57小鼠氧诱导视网膜病变的影响。方法：将新生C57 BL/6 J小鼠随机分为3组,分别为正常对照组、VAS2870注射 OIR 组和无菌 PBS 缓冲液注射OIR组。将后两组小鼠在出生后第7 d ( P7)至P12置于体积分数为75%±2%的恒定高氧氧箱中以构建OIR模型,在 P12时给予幼鼠双眼玻璃体腔注射 VAS2870(0.5μL),另一组幼鼠双眼注射同等剂量的无菌PBS缓冲液。三组小鼠均在 P17时取右眼行视网膜铺片和Lectin染色,观察视网膜中央无血管区及病理性新生血管的情况;取右眼行视网膜组织定量检测 ROS/RNS 含量;取左眼应用RT-PCR检测Nox4 mRNA含量,并应用Western-blot测定视网膜组织中VEGF的表达。结果：VAS2870注射OIR组小鼠视网膜中央无血管区面积较无菌PBS缓冲液注射OIR组明显减少( P<0.05),病理性新生血管数目明显减少( P<0.05);VAS2870注射OIR组小鼠视网膜组织Nox4 mRNA的表达量明显低于无菌PBS缓冲液注射组;VAS2870注射OIR组小鼠视网膜组织ROS含量较无菌PBS缓冲液注射组明显降低( P<0.05);VAS2870注射OIR组小鼠视网膜组织VEGF的表达明显低于无菌PBS缓冲液注射组( P<0.05)。结论：在小鼠OIR模型中, VAS2870可抑制Nox4 mRNA的表达,减少ROS/RNS,下调VEGF的生成,在视网膜病变进程中具有保护作用。     

Oxygen-induced retinopathy in the newborn rat: effects of hyperbarism and topical administration of timolol maleate

Background: Lesions resembling those of human retinopathy of prematurity can be provoked in newborn Wistar rats by exposure to an FiO₂ of 80% for the first 5 days of life followed by 5 days recovery under room-air conditions. Methods: We evaluated the effects of moderate hyperbarism (+60.75 kPa, i.e. 455 mmHg or 0.6 atm) and topical administration of 0.25% timolol maleate on oxygen-induced retinopathy (OIR) in this experimental model. Results: OIR (including neovascularization in most cases) was observed in 100% of the retinas of normobaric oxygen-reared ratlings that did not receive timolol. OIR was less frequent in oxygen-reared ratlings treated with hyperbarism (60%) or timolol (65%). Hyperbaric oxygen supplementation combined with timolol treatment during both the hyperoxic and room-air phases reduced the incidence of OIR to 30%. There was no sign of vasoproliferation in any of the retinas from the latter three groups. Conclusions: The highly significant protective effects of hyperbarism and timolol observed in this study are not fully understood. We speculate that vasoconstriction induced by the hyperbarism reduces the amount of oxygen that reaches the retina from the choroid during O₂ supplementation, while an increased ocular perfusion pressure caused by timolol-induced reduction of the intraocular pressure might decrease the stimulus to vasoproliferation that normally occurs with room-air recovery.     

Retinal vascular development and oxygen-induced retinopathy: a role for adenosine

Adenosine is a ubiquitous molecule that is produced predominantly by catabolism of adenosine triphosphate. Levels of this nucleoside increase dramatically with ischemia and elevated tissue activity. Adenosine levels are high in inner retina during retinal vascular development in postnatal dog. The source appears to be the ectoenzyme 5' nucleotidase, which is prominent at this time in the innermost process of Muller cells. One of the adenosine receptors, A(2A), is present on endothelial cell precursors, angioblasts, and endothelial cells in formed blood vessels in neonatal dog. These observations suggest that adenosine is important in retinal vascular development. Oxygen-induced retinopathy (OIR) is a model for human retinopathy of prematurity (ROP). The initial event in OIR is induced by exposure of the developing retina to high oxygen. Vascular development is halted and over 60% of the retinal vasculature is lost during this stage of the disease in dog, which is called vaso-obliteration. 5' nucleotidase is dramatically reduced during vaso-obliteration, resulting in a sharp decline in adenosine. When animals are returned to room air, the retina is hypoxic because of the lack of blood vessels, oxygen consumption is increased due to neuronal development, and systemic levels of oxygen have returned to normal. At this time, 5' nucleotidase activity and adenosine levels are elevated well beyond normal levels. This stage of OIR is the vasoproliferative stage and A(2A) expression and endothelial cell proliferation are very elevated compared to control animals. Florid preretinal neovascularization forms, which has high levels of adenosine and A(2A) receptors. Therefore, adenosine and its A(2A) receptor appear to be important in canine OIR. This work suggests that adenosine and its receptors may be a therapeutic target in OIR. This hypothesis is supported by recent studies in mouse (Mino et al., Invest. Ophthalmol. Vis. Sci. 42(13) (2001) 3320), which demonstrated that targeting one of the A(2) receptors can inhibit formation of neovascularization in OIR.     

Oxygen-induced retinopathy in the newborn rat: morphological and immunohistological findings in animals treated with topical timolol maleate

Oxygen-induced retinopathy (OIR) similar to that seen in premature infants can be produced in newborn rats by exposure to 80% oxygen for the first 5 days of life, with subsequent recovery for 7 days in a room air environment. Previous studies have shown that the latter phase is associated with degeneration of retinal astrocytes. In the absence of astrocytes, the vascular endothelial growth factor (VEGF) is produced by the neurons of the inner retina. In this study, immunohistochemical methods were used to assess retinal expression of VEGF protein in ratlings exposed to the above protocol, with and without simultaneous treatment with topical timolol maleate. Intraocular pressure (IOP) and arterial pressure were measured. The severity of OIR was also evaluated in retinal flat mounts after India ink perfusion. The mean IOP was 8.25 +/- 0.32 mm Hg in untreated ratlings and 5.15 +/- 0.24 mm Hg in timolol-treated animals. OIR in retinas from the timolol group was less severe than that seen in untreated ratlings. VEGF protein expression was almost completely absent in the latter group. Retinas from timolol-treated animals expressed VEGF protein, though the level was inferior to that found in controls raised under room air conditions. In conclusion, these data seem to indicate that experimental OIR can be attenuated by a reduction of the IOP during and after oxygen supplementation. It is possible that these effects are due to improved ocular perfusion pressure, which would mitigate the hypoxic insult to the retina during the room air recovery period.     

Oxygen-induced proliferative retinopathy in the newborn rat

The neonatal rat, which has an immature retinal vasculature at the time of birth, is a potential animal model for retinopathy of prematurity since it has an established spindle cell retinal vasoformation pattern similar to that seen in the human. To determine if proliferative oxygen-induced retinopathy can be produced in the rat, 40 newborn rat pups were exposed from birth either to air for 25 days or to an 80% oxygen environment for 10 days, followed by 15 days in air. Extraretinal neovascularization was observed in 80% (16/20) of the rat pups exposed to hyperoxia (p less than 0.001) with a bilaterality of 87.5% (14/16). Mild to moderate vitreous hemorrhage was seen in only three eyes. Mesenchymal shunt or ridge formation was not demonstrated, nor was retinal detachment.     

Oxygen-induced retinopathy in the rat. Vitamins C and E as potential therapies.

Oxygen-induced retinopathy (OIR) was produced by subjecting newborn albino rats to a 60% oxygen atmosphere for 14 days before they were killed and retinal analysis was done. The extent of OIR was measured by estimating the severity of retinal vasoobliteration in ink-perfused flat-mounted retinas. This was done with the aid of a digitizing camera and an image-analysis system designed to create binary images of the retinal blood vessels. Retinal levels of several antioxidant molecules also were measured. Alpha-tocopherol and ascorbic acid were reduced in oxygen-exposed rats by 34% and 20%, respectively, compared with room air-raised control animals. Retinal glutathione reductase, S-transferase, and peroxidase showed no differences between oxygen-treated and -untreated rats. Attempts to increase the newborn rats' retinal ascorbic acid by administering daily subcutaneous injections (5 g/kg body weight) to the mother rats were unsuccessful. However, the level of retinal alpha-tocopherol of newborn rats could be altered by dietary manipulation of the mothers. The mothers were fed diets containing either 1 g alpha-tocopherol acetate/kg food or none, starting 21-25 days before the birth of their litters and lasting throughout the exposure period. This treatment resulted in three- to fourfold differences in the retinal alpha-tocopherol levels of the pups. The combination of dietary and oxygen treatments also resulted in significant differences in retinal glutathione peroxidase activity, with the vitamin E-deprived, oxygen-exposed group having highest levels. Newborn rats both supplemented with and deprived of alpha-tocopherol had less vasoobliteration than did those nursed by mothers fed rat chow.     

改良视网膜铺片联合免疫荧光染色技术在氧诱导视网膜病变模型中的应用

背景 氧诱导视网膜病变(OIR)动物模型的视网膜铺片是缺血性视网膜病变研究的有用工具.OIR大鼠或小鼠的幼鼠视网膜面积小且厚,采用传统方法先剪开视网膜再进行铺片实际操作难度较大,影响了对实验结果的定量分析. 目的 探讨一种易操作、稳定性好的鼠视网膜铺片联合免疫荧光染色技术.方法采用随机数字表法将40只出生后＜6 h的SD大鼠随机分为OIR模型组和正常对照组,OIR模型组幼鼠与母鼠一起以24 h的时间间隔交替在体积分数80％高氧环境或21％常氧环境中喂养14d,正常对照组大鼠在常氧环境中喂养.于喂养至14 d时摘除眼球并剥离视网膜,对完整的视网膜先行谷氨酰胺合成酶(GS)-isolectin B4染色,然后展平视网膜铺片并呈放射状切为4瓣,并于荧光显微镜下拍照,应用Adobe PhotoshopCS3图像分析软件系统进行图像拼接处理,获得全视网膜血管图像,采用软件测量每张完整视网膜铺片图像中无血管区及整个视网膜的像素值,最后计算无血管区的像素百分比进行无灌注区严重程度分析. 结果采用先行视网膜血管免疫荧光染色再放射状切开视网膜的方法可见视网膜结构完整,展开的视网膜铺片平整,视网膜全周均可见锯齿缘结构.视网膜血管呈强的绿色荧光,血管分支清晰可见,而视网膜的背景绿色荧光较弱.正常对照组大鼠视网膜铺片显示其血管基本发育完全,OIR模型组大鼠视网膜铺片可显示中央视盘旁无毛细血管区及周边大片无血管区形成. 结论 采用先行视网膜血管免疫荧光染色再放射状切开视网膜的方法制备视网膜铺片简便、易行,较传统的视网膜铺片法更容易确保视网膜结构的完整性,有利于OIR模型视网膜血管形态学的观察和分析.     

抗血管内皮生长因子对氧诱导视网膜新生血管病变模型小鼠视网膜多巴胺含量的影响

目的　研究抗血管内皮生长因子（VEGF）融合蛋白康柏西普（Conbercept）玻璃体内注射对小鼠氧诱导视网膜新生血管病变（OIR）模型中视网膜多巴胺（DA）含量的影响。方法　普通级7 d龄C57BL/6小鼠100只,随机分为空白对照组、OIR组、OIR+生理盐水（NS）组及OIR+ Conbercept组,每组25只。其中空白对照组小鼠在常氧环境中饲养至17 d龄。OIR组、OIR+NS组及OIR+ Conbercept组小鼠通过高流量吸氧建立OIR模型,并在此环境中饲养至12 d龄,OIR+NS组和OIR+Conbercept组小鼠分别行右眼玻璃体内注射1 μL NS、1 μL Conbercept后,在常氧环境中饲养至17 d龄,处死。取各组小鼠右眼眼球行HE染色及视网膜铺片,观察突破内界膜的视网膜血管内皮细胞核数及血管分布;行Western blot检测各组小鼠视网膜中酪氨酸羟化酶（TH）、血管内皮生长因子（VEGF）表达量;行高效液相色谱仪检测各组小鼠视网膜DA含量。结果　空白对照组小鼠视网膜各层清晰,未见明显无灌注区及新生血管。与空白对照组相比,OIR组小鼠视网膜各层排列紊乱,视盘周围可见大片无灌注区,突破内界膜的血管内皮细胞核数、VEGF相对表达量均增加,TH相对表达量、DA含量均减少,差异均有统计学意义（均为P<0.05）;OIR+NS组与OIR组相比,小鼠视网膜中突破内界膜的血管内皮细胞核数、DA含量及VEGF、TH相对表达量差异均无统计学意义（均为P>0.05）;与OIR组及OIR+NS组相比,OIR+ Conbercept组小鼠视网膜各层排列较清晰,视盘周围可见小片状无灌注区,突破内界膜的内皮细胞核数、DA含量及VEGF、TH相对表达量均减少,差异均有统计学意义（均为P<0.05）。结论　OIR模型中视网膜DA含量及TH相对表达量均降低;玻璃体内注射Conbercept后,视网膜新生血管及VEGF相对表达量均减少,TH相对表达量、DA含量均进一步降低。     

Role of unc5b in retinal neovascularization in mice with oxygen-induced retinopathy

AIM: To explore the role of unc5b in retinal neovascularization in murine oxygen-induced retinopathy (OIR). METHODS: On postnatal 7(P7), C57BL/6J mice were exposed to 75%±2% oxygen for 5 days. On postnatal 12(P12), the mice were brought back to the room air (21% oxygen) to induce retinal neovascularization. Western blot analysis was performed to examine the temporal expression of unc5b in murine retinas. Double staining for unc5b and isolectin B4 were employed to determine the location of unc5b in murine retinas. The effect of unc5b on retinal neovascularization was evaluated by intravitreal injection of unc5b-FC in mice with OIR. Retinal neovascularization was measured by counting neovascular cell nuclei above the internal limiting membrane and by angiography of flat-mounted retinas perfused with fluorescein dextran. RESULTS: Compared to age-matched normal mice, the expression of unc5b was significantly increased in retinas of OIR mice on P17 and P21. Unc5b was apparently expressed in retinal vessels of OIR while being negative in normal retinal vessels. Retinal neovascularization in eyes injected with unc5b-FC was significantly reduced. CONCLUSION: Unc5b-FC can effectively inhibit retinal neovascularization induced by OIR. It may serve as a powerful and novel therapy for ischemia-induced retinal disease.     

A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM: To explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR). METHODS: Thirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin(GSL);(3) Immunofluore- scence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified- CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software. RESULTS: GSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovas- cularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group. CONCLUSION: GSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.     

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。