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。     

The effect of Sairei-to on oxygen-induced retinal neovascularization in the neonatal rat

PURPOSE: To study the effect of Sairei-to (ST), a Japanese traditional medicine, on oxygen-induced retinopathy (OIR) in rats. METHODS: OIR was induced by maintaining Sprague-Dawley neonatal rats in 80% oxygen for 12 days. The rats were treated once daily with oral administration of 0.75 g/kg (n = 9), 1.5 g/kg (n = 13) of ST in water, or water alone (WA, n = 13) at 5 mL/ kg body weight from day 6 to day 17. On day 18, retinal samples were collected. Retinal neovascularization (NV) was assessed by the NV score, and by the percentage of avascular area (% AVA), using a method previously reported. The number of severe retinal NV cases (NV > or = 9) was compared. The retinal vascular endothelial growth factor (VEGF) concentrations were measured with an immunoassay kit, at 0, 12, 24, 72 and 144 hours after oxygenation. RESULTS: NV score and % AVA decreased in the ST treated group compared to the WA group. However, severe NV was seen in five cases of WA and in one case of the ST treated group. Thus severe NV was inhibited significantly by ST treatment (p = 0.0185). Retinal VEGF did not differ between groups at any time points. CONCLUSION: These data suggest that severe NV in OIR is inhibited by ST treatment.     

Spatial pattern and temporal evolution of retinal oxygenation response in oxygen-induced retinopathy

PURPOSE: To determine the spatial pattern and temporal evolution of the change in retinal partial oxygen pressure (DeltaPO(2)) associated with a murine oxygen-induced retinopathy (OIR) model of retinal neovascularization (NV). METHODS: On P7, newborn C57BL/6 mice were exposed to 75% oxygen until postnatal day (P)12, followed by recovery in room air until P17 or P34. Control mice remained in room air until P17 or P34. At P17 and P34, functional magnetic resonance imaging (MRI) and a carbogen inhalation challenge was used to measure retinal DeltaPO(2). Retinal avascularity, distance from the optic nerve head to the vascular edge in the peripheral retina, and NV incidence and severity were measured in retinas stained with adenosine diphosphatase (ADPase). RESULTS: In P17 and P34 controls and in P34 OIR animals, retinas were fully vascularized without evidence of NV. In P17 OIR mice, there was a large central retinal capillary-free zone (22% +/- 3% of the entire retinal area, mean +/- SD) and 4 clockhours (range 1-7) of retinal NV at the border of the peripheral vascular and central acapillary retina in 100% (36/36) of the mice. In P17 OIR mice, retinal DeltaPO(2) over the vascularized far peripheral retina was not significantly (P > 0.05) different from the P17 control but was supernormal (P < 0.05) over the central capillary-free retina. However, no differences (P > 0.05) in retinal DeltaPO(2) were found between the P34 control and OIR groups. CONCLUSIONS: A reversible supernormal DeltaPO(2) was found only over the central acapillary retina during the appearance of retinal NV in a mouse OIR model. The present data show the applicability of carbogen-challenge functional MRI to the study of retinal DeltaPO(2) in vivo in eyes that are too small for the use of existing techniques.     

甲巯咪唑诱导的低浓度血清IGF-1对高氧所致新生大鼠视网膜新生血管的影响作用研究

目的 了解甲巯咪唑(MMI)对氧诱导的新生大鼠视网膜病变的影响,以期进一步明确低浓度血清胰岛素样生长因子-1(IGF-1)是否为早产儿视网膜病变的危险因素.方法 实验研究.新生SD大鼠96只,随机分为4组,分别为循环氧组:幼鼠出生后在循环氧条件下饲养14 d后,置于空气环境中4 d,母鼠饮用普通自来水;对照组及MMI组:幼鼠皆于空气中饲养18 d,母鼠分别饮用普通自来水和含0.1%MMI的水;"循环氧+MMI"组:循环氧组实验条件下的母鼠饮用含MMI的水.每组取右眼进行视网膜铺片和二磷酸腺苷(ADP)酶染色,左眼行石蜡切片后计数突破视网膜内界膜的细胞核数目,对视网膜血管进行评估;用放射免疫分析法进行血清IGF-1的测定.同时观察各组新生鼠体重的变化.采用x2检验方法比较各组新生血管发生率,成组t检验或单因素方差分析比较各组视网膜无血管区占整个视网膜面积的比值、视网膜血管密度、内界膜前血管内皮细胞核计数和各组间血清IGF-1浓度.结果 循环氧组同"循环氧+MMI"组相比,NV的发生率分别为26%和57%(x2=4.38,P=0.04);视网膜内界膜前血管内皮细胞核计数分别为(33.17 ±3.06)和(65.64±3.85)(t=17.73,P=0.00),差异均有统计学意义;"循环氧+MMI"组视网膜无血管区占整个视网膜面积的6.37%±1.23%,其余各组视网膜血管发育至周边部;对照组、MMI组、循环氧组及"循环氧+MMI"组间视网膜血管密度分别为95.21±6.17、52.57±4.14、68.82±3.71、64.20±4.26.差异有统计学意义(F=331.74,P=0.00);血清IGF-1含量分别为(536.43±32.65)、(235.94±29.09)、(526.50±26.83)、(227.24 ±19.59)mg/L,差异有统计学意义(F=278.57,P=0.00).饮用普通自来水的对照组和循环氧组幼鼠生长发育基本正常,而饮用MMI的MMI组和"循环氧+MMI"组幼鼠可见明显的生长发育迟缓.结论 研究显示MMI能加重高氧诱导的新生大鼠视网膜新生血管的发生率和严重程度.低浓度血清IGF-1可能是非氧相关调节中重要的因素之一,其调控新生血管形成的机制有待于进一步研究.     

The anti-thyroid drug methimazole induces neovascularization in the neonatal rat analogous to ROP

PURPOSE: To determine the effect of methimazole (MMI), an anti-thyroid drug known to reduce serum l-thyroxine (T4), and insulin-like growth factor (IGF)-1 concentrations, on retinal vascular development in neonatal rats. METHODS: Sprague-Dawley rats (n=175) were raised in expanded litters of 25 in room air and were exposed to MMI from birth (given as a 0.1% solution to nursing mothers for either 4 or 10 days). Experiments ended on day 4 (n=25) or 10 (n=50) of life. A third group was exposed to MMI for the initial 4 days of life and then allowed to recover for the next 6 days (n=50). Fifty control rats were analyzed on day 4 (n=25) or 10 (n=25) of life. Left eyes were fixed, and retinas were dissected and stained with adenosine diphosphatase (ADPase). Retinas were graded for presence and severity of neovascularization (NV) in a masked manner, and retinal vascular areas were quantified. In a subsequent study, serum IGF-1 and T4 levels were measured by radioimmunoassay in an additional 200 rats exposed to treatments identical to those described. RESULTS: Retinal NV occurred in 31% of rats exposed to 10 days of MMI and 4% (P=0.02) of rats exposed to 4 days of MMI, followed by 6 days of recovery. None of the rats exposed to 4 days of MMI alone and none of the control animals was graded positive for NV. Retinal vascular areas were significantly reduced in rats exposed to 4 days of MMI compared with 4-day control animals (36% +/- 6% vs. 50% +/- 6%, P=0.0001). Serum IGF-1 levels were markedly reduced in 4-day MMI rats compared with age-matched control animals (42 ng/mL vs. 133 ng/mL, P=0.0001) and in 10-day MMI rats compared with 10-day control animals (133 ng/mL vs. 206.5 ng/mL, P=0.005). Serum T4 levels were similarly suppressed in the MMI-exposed litters compared with control animals at day 10 (P=0.008). In contrast, rats exposed to 4 days of MMI followed by 6 days of recovery had normal serum IGF-1 and T4 levels by day 10. CONCLUSIONS: The anti-thyroid drug, MMI, induces NV in neonatal rats. This may be mediated by the initial suppression of serum IGF-1. Nevertheless, the lower incidence of NV when serum IGF-1 levels are initially suppressed followed by complete recovery, is contrary to a purely permissive role for serum IGF-1, as reported previously. The relationship between the temporal course of serum IGF-1 and NV in immature retinas needs further investigation.     

Suppression of retinal neovascularization by the iNOS inhibitor aminoguanidine in mice of oxygen-induced retinopathy

Background  Retinal neovascularization (NV) is a major cause of blindness associated with ischemic retinal disorders. Our study was focused on evaluating the inhibitory effect of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), on retinal NV in mice of oxygen-induced retinopathy (OIR). Methods  An OIR model was established with 7-day-old C57BL/6J mice. One day before and 1 and 3 days after being returned to the room air, the right eyes were injected intravitreally with bevacizumab, AG or bevacizumab+AG respectively. The left eyes were injected with normal saline (NS) as control. The mice were killed at postnatal day 17 (P17). The effects of AG or bevacizumab on iNOS or VEGF expressions were evaluated by RT-PCR and immunohistochemistry. Retinal NV was examined by fluorescein angiography, and was quantified histologically by CD34 immnunostaining at P17. Results  Compared with NS-treated eyes, retinal VEGF and iNOS mRNA expressions were significantly reduced in AG- and bevacizumab+AG-treated eyes; whereas in bevacizumab-treated eyes, retinal VEGF mRNA expression increased and iNOS mRNA expression remained unchanged. The above changes were confirmed by immunohistochemical study. The generalized decrease in both VEGF and iNOS distributions in mice retina treated with AG or bevacizumab+AG was demonstrated by immunohistochemistry. Retinal NV was significantly reduced in all three groups treated with bevacizumab, AG or bevacizumab+AG, when compared with NS-treated eyes. Conclusions  iNOS activation plays a pathological role in retinal NV in a mouse model of ischemic retinopathy. Administration of AG significantly suppressed retinal NV. Therefore, AG appears to be a novel and effective therapeutic approach for retinal NV. Ling Wang and Guo-Tong Xu are co-corresponding authors who contributed equally to this study.     

Neovascularization grading methods in a rat model of retinopathy of prematurity

PURPOSE: The method of counting cell nuclei above the internal limiting membrane in histologic sections is considered the standard when quantifying neovascularization (NV) in rodent oxygen-induced retinopathy (OIR). An alternative, more rapid method of counting clock hours in flatmounted adenosine diphosphatase (ADPase)-stained rat retinas is analogous to clinically scoring retinopathy of prematurity (ROP). In the present study, the validity of counting clock hours was evaluated by a direct comparison of these techniques. The intereye correlation of NV score and retinal vascular area were also studied. METHODS: Newborn Sprague-Dawley rats were exposed to cycles of O2 (80-10%) for 7 days, followed by 5 days of room air recovery. Preretinal NV was quantified by three masked observers counting clock hours in flatmounted ADPase-stained retinas of both eyes. Retinal vascular and total retinal areas were calculated using computer-assisted analysis. Representative retinas that had been scored positive (n = 10) and negative (n = 3) for NV and room air control retinas (n = 3) were embedded in paraffin. Each entire peripheral retinal quadrant was serially sectioned at 6 microm and stained with hematoxylin and eosin. Nuclei above the internal limiting membrane were then counted in a masked manner. The total number of nuclei counted per retina was defined as the nucleus count (704-938 sections per retina; 12,900 sections). Correlations were evaluated using Spearman rank coefficients. RESULTS: The nucleus count was 0 to 44 in room air control retinas, 0 to 40 in negative OIR retinas, and 250 to 5634 in positive OIR retinas. The nucleus count was highly correlated with the clock hour score (r(s) = 0.95, P = 0.0001). For the paired retinas, there was a significant correlation between right and left eyes in the severity of NV (clock hours; r(s) = 0.76, P = 0.0001) and the ratio of retinal vascular area to total retinal area (r(s) = 0.81, P = 0.0001). CONCLUSIONS: The more rapid method of counting clock hours in flatmounted ADPase-stained retinas is valid for quantifying NV in rat models of ROP. Incidence and severity of NV and vascularized areas were similar between left and right eyes, which permits the use of paired retinas for complementary research techniques.     

Activated NAD(P)H oxidase from supplemental oxygen induces neovascularization independent of VEGF in retinopathy of prematurity model

PURPOSE: To study NAD(P)H oxidase-dependent outcomes after oxygen stresses that are similar to those experienced by preterm infants today using a rat model of retinopathy of prematurity. METHODS: Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days and were returned to room air (21% O2, 50/10 oxygen-induced retinopathy [OIR]) or supplemental oxygen (28% O2, 50/10 OIR+SO) for 4 days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor apocynin (10 mg/kg/d) or of PBS from postnatal day (P)12 to P17, and some received intraperitoneal injections of hypoxyprobe before kill. Intravitreous neovascularization (IVNV), avascular/total retinal areas, vascular endothelial growth factor (VEGF), NAD(P)H oxidase activity, or hypoxic retina (conjugated hypoxyprobe) were determined in neurosensory retinas. Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or control were exposed to 1% or 21% O2 and assayed for phosphorylated (p-)Janus kinase (JNK) and NAD(P)H oxidase activity. RESULTS: Retinas from 50/10 OIR+SO had increased NAD(P)H oxidase activity and lower VEGF than did retinas from 50/10 OIR. Apocynin treatment reduced the IVNV area and hypoxic retina in 50/10 OIR+SO. RMVECs treated with 1% O2 had increased p-JNK compared with RMVECs exposed to room air. CONCLUSIONS: Different oxygen stresses activate NAD(P)H oxidase to varying degrees to trigger disparate pathways (angiogenesis or apoptosis). The oxygen stresses and outcomes used in this study are relevant to human ROP and may explain some of the complexity in the pathophysiology of ROP resulting from oxygen exposure.     

糖尿病鼠视网膜细胞间黏附分子-1的表达与血视网膜屏障破坏的关系及曲安奈德的治疗作用

目的 探讨糖尿病(DM)鼠视网膜血管细胞间黏附分子-1(ICAM-1)在血视网膜屏障(BRB)破坏中的作用及玻璃体注射曲安奈德(TA)对BRB破坏的治疗作用。〓 方法 60只Wistar鼠建立DM模型，分为对照组、DM 4个月组和DM 6个月组。每组各分为免疫组织化学组和BRB测定组，BRB测定组再分为未行TA治疗组、TA治疗1周组和2周组。大鼠玻璃体内注射TA 5μl。视网膜铺片免疫组织化学染色观察视网膜ICAM-1的表达及形态学变化，图像分析软件测定内皮细胞平均吸光度(A)［旧称光密度（OD）］，定量ICAM-1表达。测定视网膜伊凡思蓝(EB)含量，评价BRB的变化。 结果 免疫组织化学组:对照组视网膜血管无明显ICAM-1阳性表达，同对照组相比，DM 4个月组阳性表达显著增强(P＜0.001)，已出现毛细血管管径粗细不一等形态学改变；DM 6个月组阳性表达进一步增强(P＜0.001)，形态学改变进一步加重，可出现无细胞性毛细血管。BRB测定组:各对照组间EB含量无统计学差异(P＞0.05)。未行TA治疗组中，两DM组EB含量明显高于对照组(P＜0.001)，且DM 6个月组高于4个月组(P＜0.01)。TA治疗组中，各DM组EB含量均显著降低(P＜0.001)，但组间无统计学差异(P＞0.05)，然而DM 4个月治疗2周组已基本恢复正常(P＞0.05)，余治疗组仍高于对照组(P＜0.05)。内皮细胞 A 值与视网膜EB含量呈直线相关(r=－0.959)。 结论 糖尿病鼠视网膜ICAM-1的表达与BRB破坏呈正相关。玻璃体内注射TA可有效减轻BRB破坏。 (中华眼底病杂志, 2006, 22: 24-27)     

Triamcinolone Acetonide Prevents Enhancement of Hypoxia-induced Neuronal and Inducible Nitric Oxide Synthases in the Retinas of Rats with Oxygen-induced Retinopathy

Purpose

We investigated whether oxygen-induced retinopathy (OIR) results in changes in the protein expression of neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectively) in rat model of OIR. In addition, we evaluated whether treatment of rats with triamcinolone acetonide (TA) prevents this response.

Methods

To promote OIR, Sprague-Dawley rats were exposed to hyperoxia from postnatal day 2 (P2) to P14. They were then returned to normoxia after P15. TA was injected into the right vitreous of P15 rats, while saline was injected into the left vitreous. At P18 the expression of nNOS and iNOS was determined using Western blotting and immunostaining techniques in retinas obtained from control rats.

Results

In P18 OIR rats, the abundance of nNOS and iNOS protein was significantly increased compared with controls. These increases were not observed in the retinas of rats treated with TA. The change in expression of nNOS and iNOS were specific to parvalbumin and glial fibrillary acidic protein-positive cells. Treatment with TA prevented the increased expression of nNOS and iNOS in all samples.

Conclusions

Hypoxia upregulates expression of nNOS and iNOS in OIR rat retinas, which is can be prevented by treatment with TA.     

Expressions of survivin and vascular endothelial growth factor in a Murine model of proliferative retinopathy

AIM: To examine the expression of survivin and vascular endothelial growth factor(VEGF) during the development of retinal neovascularization (NV) in a mouse model. METHODS: A well-characterized murine model of retinal NV was used to study the expression of survivin and VEGF. NV of the retina was induced in mice by exposure to 75% O2 from postnatal day P7 to P12, followed by return to room air from P12 to P17. Expression of survivin and VEGF protein was analyzed by Immunohistochemistry. In addition, mouse model of proliferative retinopathy was analyzed by retinal fluorescein angiography and quantification analysis. RESULTS: The normal mice had both superfiekal and deep vascular layers that extended from the optic nerve to the periphery. In intraocular pressure(IOP) mice were characterized by represent a typical pattern of pathological retinal NV. There are less or little nuclei of new vessels vascular endothelial cell breaking through the inner retinal than in retinopathy of prematurity (ROP) mice, large clusters of blood vessels were adherent to the internal limiting membrane(ILM) (0.27±0.20 vs 23.38±1.027, t=9.454, P<0.001). During the angiogenic period from P13 to P17, survivin and VEGF protein expression increased in experimental retinas compared with control samples(2.56±0.46 vs 3.34±0.40, t=17.43, P<0.01: 2.18±0.75 vs 4.34±0.25, t=19.61, P<0.01). Protein levels of VEGF and survivn has significantly positive correlation (P<0.05, r=0.411). CONCLUSION: Correlation was made at the protein levels of survivin expression compared with that of VEGF in a murine model of retinal NV, which suggests a temporal role for survivin and VEGF in new vessel formation in response to hypoxic stimulation.     

Attenuation of EphrinB2 Reverse Signaling Decreases Vascularized Area and Preretinal Vascular Tuft Formation in the Murine Model of Oxygen-Induced Retinopathy

Purpose. EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. Methods. Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. Results. EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. Conclusions. EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.     

Captopril and vascular endothelial growth factor in a mouse model of retinopathy

PURPOSE: Angiotensin converting enzyme (ACE) inhibition has been shown in animal models of retinopathy and in patients with diabetes to improve retinal neovascularization. The mechanism is not clearly identified, but could potentially be mediated via vascular endothelial growth factor modification. The objective of this study was to determine the effect of captopril, an angiotensin converting enzyme (ACE) inhibitor, on retinal VEGF, VEGF-R1, and VEGF-R2 expression in a mouse model of oxygen induced retinopathy (OIR). METHODS: A mouse model of OIR was used and retinal tissue was obtained at P7, prior to oxygen exposure, at P12, just after oxygen exposure, and at P17, the time of maximal retinal neovascularization for VEGF, VEGF-R1 and VEGF-R2 assessment. A group of animals were treated with captopril (0.5 mg/kg/d SC from P7 for five days). RESULTS: Captopril plus OIR treated animals had higher levels of retinal VEGF mRNA and protein at P12 (p < 0.05) and lower levels at P17 (p < 0.05) than OIR animals. VEGF-R1 mRNA expression increased 16 fold from P7 to P17 (p < 0.05) in room air reared animals. VEGF-R1 mRNA expression was unaffected by OIR and/or captopril treatment. VEGF-R2 mRNA expression decreased from P7 to P17 by 1.5-fold in room air reared animals (p = 0.001). Retinal VEGF-R2 mRNA and protein expression were significantly higher at P12 in OIR plus captopril treated animals than OIR animals (p = 0.01). CONCLUSIONS: In summary, captopril maintains VEGF and increases VEGF-R2 expression during the period of hyperoxia when VEGF expression is normally suppressed. Captopril treatment during oxygen exposure is associated with a reduction in the angiogenic response at day 17 as manifested by decreased VEGF and VEGF-R2 expression in retinal tissue. Angiotensin converting enzyme inhibition is associated with changes in expression of VEGF and VEGF-R2 in the evolution of retinal neovascularization in the mouse model of retinopathy.     

CD147、基质金属蛋白酶-2和血管内皮生长因子在氧诱导视网膜病变大鼠中的表达

目的 观察CD147、基质金属蛋白酶（MMP）-2和血管内皮生长因子（VEGF）在氧诱导视网膜病变大鼠中的表达。方法 84只新生Wistar大鼠纳入研究。将其随机分为高氧组和对照组。每组42只大鼠。高氧组建立氧诱导视网膜病变模型;对照组不做任何处理。大鼠7、14日龄时,取眼球行视网膜铺片,观察视网膜血管形态。大鼠14日龄时,取眼球行视网膜石蜡切片,计数突破内界膜的内皮细胞核数。大鼠12、14、16日龄时,取眼球行免疫组织化学染色,检测大鼠视网膜组织中CD147、MMP-2和VEGF的表达。采用双因素相关分析法分析CD147表达与MMP-2、VEGF表达的相关性。结果 大鼠7日龄时,对照组大鼠视网膜血管呈放射状分布,血管径较粗;高氧组大鼠视网膜血管收缩,大面积无血管区,仅见少量小面积的血管网。大鼠14日龄时,对照组大鼠视网膜血管发育较好,网格规整;高氧组大鼠视网膜血管增生、纡曲、扩张,并在灌注区与非灌注区交界处形成新生血管丛、渗出、出血。大鼠14日龄时,对照组、高氧组突破内界膜的内皮细胞核数分别为（1.30±1.26）、（19.70±3.56）个;两组比较,差异有统计学意义（t=21.813,P＜0.01）。免疫组织化学染色结果显示,对照组大鼠视网膜各层可见CD147、MMP-2、VEGF有少量表达;高氧组大鼠视网膜各层可见CD147、MMP-2、VEGF有大量表达。大鼠12、14、16日龄时,两组大鼠视网膜各层中CD147（t=5.612、11.390、6.355）、MMP-2（t=4.122、8.047、4.422）、VEGF（t=4.955、12.176、5.110）表达比较,差异均有统计学意义（P＜0.01）。相关性分析发现,CD147的表达与MMP-2、VEGF的表达均呈正相关(r=0.755、0.820,P＜0.01）。 结论 CD147、VEGF、MMP-2在氧诱导视网膜病变大鼠中有大量表达。     

Evaluation of toxicity due to vital stains in isolated rat retinas

PURPOSE: We investigated whether artificial aqueous humours are adequate incubation media compared with artificial cerebrospinal fluid (aCSF), and evaluated the retinal toxicity of two vital stains--trypan blue (TB) and indocyanine green (ICG)--and triamcinolone acetonide (TA) using isolated rat retinas incubated in artificial aqueous humours. METHODS: In experiment 1, retinal segments were isolated and incubated in aCSF, BSS plus, Opeguard Neo Kit, or phosphate-buffered saline (PBS). In experiment 2, retinal tissues were exposed to one of the agents and incubated in BSS plus. Retinal damage was assessed by morphological examination and biochemical assay, which measured lactate dehydrogenase (LDH) in the medium once every hour. RESULTS: In experiment 1, BSS plus was confirmed as a suitable incubation medium. In experiment 2, there were no significant changes in the retinas exposed to TB or TA. Tissues exposed to ICG showed damage in every retinal layer and significantly higher release of LDH. CONCLUSION: Exposure to ICG caused retinal damage in isolated rat retina tissue in our experimental model (in vitro).     

Effect of VEGF trap on normal retinal vascular development and oxygen-induced retinopathy in the dog

Purpose. To evaluate the effects of a vascular endothelial growth factor trap (VEGF Trap) on retinal vascular development and pathologic neovascularization (NV) in the canine model of oxygen-induced retinopathy (OIR). Methods. Newborn dogs (postnatal day [P]1) were exposed to 100% O(2) and then returned to room air on P5. VEGF Trap (5, 25, or 250 μg) was injected intravitreally in one eye and human FC (hFc) injected in the fellow eye of air control and oxygen-treated dogs on P8. The retinal vasculature and NV were evaluated on P21. Other oxygen-exposed animals received 5 μg of VEGF Trap or hFc on P22 after confirmation of retinopathy of prematurity (ROP)-like pathology and were evaluated at P45. Results. In air controls, both the vascularized area of the retina and the density of superficial capillaries were reduced in 250 or 25 μg VEGF Trap-injected eyes, and deep capillaries were absent. Eyes that received the 5 μg dose were indistinguishable from controls. In oxygen-treated animals, all eyes injected with VEGF Trap exhibited markedly less intravitreal NV than that of hFc-injected fellow eyes, irrespective of dose. Retinal vascular area in OIR animals was significantly reduced in eyes injected with 250 or 25 μg of VEGF Trap, but the 5 μg dose did not inhibit retinal revascularization. Eyes with existing NV that received 5 μg VEGF Trap at P22 exhibited substantial resolution of OIR pathology at P45. Conclusions. The VEGF Trap inhibited the formation of NV, but higher doses also inhibited revascularization of retina when injected at P8. In contrast, the lowest dose tested effectively blocked NV and caused regression of existing NV, without appreciably affecting vasculogenesis or retinal revascularization. These findings suggest that dose selection is an important variable when considering the use of VEGF-targeting agents for the treatment of ROP.     

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.