【摘要】目的：观察精氨酸－谷氨酰胺（ArgGln）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ArgGln（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。Realtime RTPCR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：ArgGln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。
【关键词】 视网膜新生血管；精氨酸谷氨酰胺；早产儿视网膜病变;氧诱导视网膜病变
Abstract
AIM: To investigate the effect of the dipeptide ArgGln on retinal neovascularization of retinopathy of prematurity (ROP) in the oxygeninduced retinopathy(OIR) animal model.METHODS: Fortyeight 7dayold C57BL/6J mice were exposed to 750mL/L oxygen for 5 days and then to normal situation to produce the murine model of oxygeninduced retinopathy(OIR). All mice received twice daily intraperitoneal injections of PBS or the dipeptide ArgGln(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 ArgGln, 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 realtime RTPCR.RESULTS: Neovascularization reduced in retinas of the dipeptide ArgGln treated group in a dosedependent manner. Compared with control group, experimental group had diminished nonperfusion 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. ArgGln at 5g/kg per day reduced preretinal neovascularization by about 75%(P<0.01). There was a significant reduction in VEGF mRNA at the 17th day in ArgGln treated group compared with control group(P＜0.01).CONCLUSION: ArgGln 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.
KEYWORDS: retinal neovascularization; arginineglutamine; retinopathy of prematurity; oxygeninduced retinopathy
INTRODUCTION
With improved survival of very lowbirthweight infants in China, retinopathy of prematurity (ROP) is emerging as a significant problem which is the major cause of blindness in children at present[1]. The common pathologic features of ROP is ischemiainduced retinal neovascularization. These changes lead to hemorrhage, tissue damage, and retinal scarring, which ultimately lead to vision loss and blindness. At present, retinal laser photocoagulation and cryotherapy appear to be the most effective treatment for retinal neovascularization[2,3]. However, these procedures can destroy postmitotic retinal neurons and permanently affect visual function. It is important to find safe preventive measures or early intervention modalities of treatment for ROP. In this study, we examined the effect of ArgGln on animal models of ROP.
MATERIALS AND METHODS
Mouse Model of Oxygeninduced Retinopathy Mouse model of oxygeninduced retinopathy(OIR) was produced in C57BL/6J mice by a method described by Smith et al[4]. Fortyeight sevendayold(postnatal day 7)mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of (750±50)mL/L oxygen for 5 days. incubator temperature was maintained at (23±2)℃. Then they were returned to room air at postnatal day 12. At the twelfth day 36 mice in experimental group received twice daily intraperitoneal injections of the dipeptide ArgGln(1.0, 3.0, 5.0g/kg per day, each group consists of 12 mice), starting on postnatal day 12 and continuing through till postnatal day 17, and 12 mice in control group received twice daily intraperitoneal injections of PBS at the same time.
Observation of Retinal Neovascularization by ADPase Histochemical Technique On the 17th day, 8 mice(2 mice in each group) were anesthetized with either an intramuscular injection of ketamine (80mg/kg) and xylazine (15mg/kg).To evaluate vessel morphology, all eyes were removed and fixed with 40g/L paraformaldehyde in phosphatebuffered saline overnight. The cornea, lens, and vitreous were surgically removed and retinas were dissected. Retinas were processed for magnesiumactivated adenosine diphosphatase (ADPase) staining as previously described by Lutty and McLeod[5]. ADPasestained retinas were flatmounted on microscope slides with gelatincoated cover slips. The vasculature was then examined under microscopy.
Quantification of Retinal Neovascularization On the 17th day, 12 mice (3 mice in each group) were sacrificed and the eyes were enucleated, immersed in 40g/L paraformaldehyde in PBS for at least 24 hours, and embedded in paraffin. Serial 6μm sections from whole eyes were cut sagittally parallel to the optic nerve and stained with hematoxylin and eosin according to a standard protocol. The extent of neovascularization was determined by counting neovascular cell nuclei extending through the internal limiting membrane into the vitreous. All counting was done based on a masked protocol. For each eye, 10 intact sections of equal length, each 30μm apart, were evaluated. The mean number of neovascular nuclei per section per eye was then determined.
VEGF mRNA Detected by RTPCR On the 17th day, 28 mice (7 mice in each group) were sacrificed and retinas were then dissected from the eye. Total RNA was isolated from mouse retina (TRIzol reagent; Invitrogen) according to the manufacturers protocol. The cDNA was synthesized by using 2μg of total RNA and reverse transcription reagents (DRR037,TakaRa) in a 50μL RT reaction. Realtime PCR analysis was applied with 1μL cDNA per reaction(SYBR PrimeScript RTPCR kit DRR041,TakaRa). At the end of the PCR cycle, a dissociation curve was generated to ensure the amplification of a single product, and the threshold cycle time (Ct) for each gene was determined. Relative mRNA levels were calculated based on the Ct and normalized to βactin. The level of VEGF mRNA determined for the injection of PBS was set to 100%. These experiments were performed using the mouse VEGF primer pair and the primer pair (D3751βactin internal standards,TakaRA).
Statistical Analysis All data were represented as the mean±SD. ANOVA was used to evaluate differences among groups. P＜0.05 was considered significant.
RESULTS
ADPase Histochemical Technique in Retinal Flatmounts The pattern of vascular development and neovascularization was seen readily in retinal flatmounts by ADPase histochemical technique. The retinal vascular patterns in the mice of control group were characterized by the neovascular tufts and avascular area,a typical pattern of pathological retinalneovascularization. The retinal vessels from the ArgGlntreated mice had reduced avascular area and neovascular tufts in a dosedependent manner compared with control group (Figure 1).
Tissue Slice of HE Staining Five days of treatment of mice with various doses of ArgGln (1.0, 3.0, and 5.0g/kg per day) resulted in a significant reduction in preretinal nuclei. At the highest dose tested, there was an about 75% reduction. All dipeptidetreated samples showed significant decreases in preretinal nuclei compared with control group(P＜0.01) (Figure 2).
Effect of ArgGln Administration on VEGF mRNA in OIR Mice There was a significant reduction in VEGF mRNA at the 17th day in ArgGln treated group in a dosedependent manner compared with control group (P＜0.01). At the dose of 5.0g/kg per day, there was about 68% reduction in the level of VEGF mRNA (Figure 3).
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