作者：荆国利,高晓唯,任兵,肖云
作者单位：832000中国新疆维吾尔自治区石河子市，石河子大学医学院研究生院；830011中国新疆维吾尔自治区乌鲁木齐市，解放军474医院全军眼科中心
【摘要】目的:评价Bevacizumab抑制大鼠角膜新生血管的效果。
方法:碱烧伤诱导角膜新生血管模型，对20只wistar大鼠40眼随机平均分4组。角膜碱烧伤后分别给予结膜下注射药物溶剂和不同剂量Bevacizumab。比较并计算角膜新生血管生长面积。16d后角膜标本行HE检验和免疫组织化学检测VEGF的表达。
结果:各用药组与对照组新生血管面积比较差异有统计学意义；治疗组间新生血管面积比较差异没有统计学意义。组织学发现各药物治疗组炎性细胞浸润、新生血管形成均明显轻于对照组。新生血管对照组VEGF染色明显增强，药物注射组表达明显减弱。
结论:结膜下注射一定浓度的Bevacizumab对大鼠角膜碱烧伤形成的新生血管有抑制作用。
【关键词】 Bevacizumab 角膜新生血管 血管内皮生长因子
INTRODUCTION
Many corneal diseases, such as corneal chemical burns, thermal burns, infections and autoimmune diseases are associated with corneal neovascularization(CNV). CNV is necessary for wound repaired, damaged tissue reconstruction and growth. But excessive corneal angiogenesis will lead to corneal dissolved, transplant rejection and loss of vision. Regulation of CNV is conducive to maintaining transparent cornea and immune privilege. The studies found that the VEGF plays a major role in the CNV. The aim of this study was to evaluate the effect of the subconjunctival bevacizumab(Avastin) on the experimental induced CNV in rats induced by alkali burns.
MATERIALS AND METHODS
Experimental Animals The total of 20 adult male Wistar rats(40 corneas) , weight (180±10)g, purchased from the Experimental Animal Center of Xinjiang Medical University.
Methods Under general anaesthesia(induced by an intraperitoneally administered 400mg/kg body weight 100g/L chlora hydrate) supplemented by topical anaesthesia(4g/L Oxybuprocaine) and tropicamide, and after a conjunctival sac washing with 1∶2000 gentamicin in normal sodium, both corneas of each animal was gently placed a 2mmdiameter filter paper saturated with 1mol/L NaOH and left for 40 seconds. Then the filter paper was removed, cornea and conjunctival sac were carefully rinsed with normal sodium for 1 minute.The experiment was performed according to the Chinese Ophthalmologist Association animal care regulation and approval of the local animal research ethics committee.
Just after the cauterization, the rats were randomly divided into four groups( group have 5 rats and 10 corneas):Group 1(n=10)received a subconjunctival injection of 0.02mL of 9g/L saline solution; Group 2(n=10)received a subconjunctival injection of 0.02mL(0.5mg)of bevacizumab; Group 3(n=10)received a subconjunctival injection of 0.04mL(1.0mg) of bevacizumab; Group 4(n=10)received a subconjunctival injection of 0.08mL(2.0mg) of bevacizumab; and applied the loxacin ophthalmic ointment to the eyes. The rats were backed into the cage after recovery.
Observation of Morphology The condition of corneal opacity, corneal neovascularization, anterior chamber response and conjunctival congestion were observed by slit lamp microscope. Observation period followed 16 days after alkali burn. Length and amount of CNV developed from the limbus were measured and photographed. Reference formula: A=C/12×3.1416[r2(rl) 2], C for the CNV number of clock hours of limbus involved, the longest but least continuous curvature CNV towards the center of cornea was recorded as L, r for corneal radius [1].The CNV area of all animals at the postoperative 3,7,14days were calculated.
Histopathology At 16th day after alkali burns, the rats were deeply anesthetized and sacrificed, then enucleation was performed after the rats were killed, and the cornea of the globe was dissected with 1mm sclera. The corneas prepared for examination were immersed in 40g/L formaldehyde. After fixation for 24 hours,the samples were dehydrated, then the corneas were cut in central, embedded in paraffin with cutting surface down, cut continuously 5μm sections. Each eye prepared for 20 pathological sections and 10 randomly selected slices for staining with H＆E for light microscopy.
Immunohistochemistry (SP method) Antibododies and Reagents: mouse antihuman VEGF monoclonal antibody as primary antibody, and biotinylated goat antimouse IgG as secondary antibody. SP labeling kits were purchased from Maixin Biotechnology Inc, Fuzhou, China.
Methods All the steps followed the specification of the kits. Sections were processed with 0.01mol/L phosphate buffer solution (PBS) instead of the primary antibody, which were used as negative control.
At the 16 days after alkali burns, the corneas were obtained and fixed in 40g/L formaldehyde for 24 hours, which were cut in 5 μm thickness and dried at 60℃ for 1 hour. The samples were deparaffinized and rehydrated. Block endogenous peroxidases by soaking slides in 50μL the reagent A for 20 minutes at room temperature, then wash 3×5 minutes with PBS. Add 50μL the reagent B to each section for 20 minutes at room temperature, the redundant liquid were verted. Add 50μL the primary antibody and incubate 1 hour, then overnight at 4℃ in the frige. Add 50μL the secondary antibody to each section for 20 minutes at room temperature and wash slides with PBS. Add 50μL biotin streptavidin to each section for 20 minutes at room temperature and wash slides with PBS. Add 50μL the DAB immediately to slides and wait for color change (approximately 5 minutes).Immediately dehydrated in ethanol. Three slices were selected randomly for every eye. At 400 magnification, under the light microscopy, the number of immunofluorescent cells were counted in a single sample with five times, and the results were averaged.
Statistical Analysis SPSS13.0 for windows statistical software was used in this study. All the experimental data were expressed by Mean±standard deviation, Oneway analysis of variance (ANOVA), LSDtest was employed to compare the differences between groups. The P valve<0.05 indicated the statistical difference between two groups.
RESULTS
Morphology One day after alkali burns, the ciliary congestion were obvious in all animal eyes, the cornea center were edema and not clear. At 3rd day, the new vessels began to grow from limbus. The growth rate of CNV was the fastest from 7th day～10th day. After 10th day, the rate of CNV growth began to slow down. In 14th day, the CNV grow slimmer, some of which degenerated. As table 1 showed, in the bevacizumabtreated eyes, there were less corneal neovas cularization than in the control eyes at 7th day after alkali burns. The vascular area of the treated group was statistical differences from the control group. No adverse effects related to bevacizumab injection were observed in all treated animals.
Table 1 The area of corneal neovascularization induced by alkaliburn in different days（略）
aThe treated group was statistical differences compared with the control group in the 7th day（P＜0.05）；cThe treated group was statistical differences compared with the control group in the 14th day（P＜0.05）
Table 2 The number of VEGF positive cells in corneal stroma induced by alkaliburn at 16th days（略）
aThe treat group was statistically different compared with the control group in the 16th day（P＜0.05）
Pathological Under light microscope, superficial substantia propria layer in bevacizumab treated group had lighter edema and less inflammatory cells infiltration vs. control group. The amount of CNV in the bevacizumab treated groups was smaller, with slimmer lumens and less arrangement. (see Figure 2)
Immunohistochemistry In the eyes of control group, the endothelium and the stroma of the cornea had inflammatory cells infiltration, and VEGF positive expression, which contained larger diameter mature CNV, and the endothelium of vessel wall had positive staining. In the bevacizumabtreated group, corneal epithelium had no inflammatory cells, a few inflammatory cells scattered in the corneal stroma, and cytoplasmic VEGF was positive expression. The number of VEGF positive cells in corneal stroma were decreased in treat group compared with the control group, and the difference was statistically significant, but there is no statistically significant difference between drug intervention group (see Table 2 and Figure 3).
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