阿柏西普联合妥布霉素地塞米松抑制碱烧伤后大鼠角膜新血管形成的实验研究

Experimental study on the inhibition of aflibercept combined with tobramycin dexamethasone on corneal neovascularization in rats after corneal alkali burn

目的探讨阿柏西普联合妥布霉素地塞米松抑制碱烧伤后大鼠角膜新血管形成(CNV)的作用。方法选取无特定病原体(SPF)级6~8周龄SD大鼠40只,采用随机数字表法分为4组,每组各10只。其中,A、B、C 3组制作大鼠碱烧伤模型。A组,大鼠结膜下注射2 mg/0.05 mL阿柏西普,联合妥布霉素地塞米松滴眼液滴眼14 d,4次/d;B组,妥布霉素地塞米松滴眼液滴眼14 d,4次/d;C组,模型对照组,未行任何治疗。D组,正常大鼠,不作处理。造模后第3、7、14天,观察测量角膜厚度并计算CNV占全角膜面积的百分比。造模后第14天,处死全部大鼠,通过苏木精-伊红染色法(HE)和免疫组化观察角膜组织中血管内皮生长因子(VEGF)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)蛋白表达水平,以及通过酶联免疫吸附法(Elisa)检测各组VEGF的蛋白浓度。结果造模后第3天,A组、B组、C组CNV面积占比分别为2.97%(2.46%,4.15%)、3.00%(1.97%,6.37%)、5.67%(4.25%,7.36%),A组、B组显著低于C组,差异均有统计学意义(t=-2.72、-2.08,P=0.007、0.037);造模后第7天、第14天,A组CNV面积占比为5.06%(3.28%,6.43%)、4.95%(3.02%,7.00%),均显著低于B组的7.83%(5.61%,9.65%)、8.64%(6.48%,10.15%)和C组的7.97%(6.42%,12.32%)、10.15%(6.26%,15.31%),差异均有统计学意义(均P<0.05)。造模后第3天,A组、B组、C组角膜厚度依次为(302.00±21.73)、(294.80±15.23)及(311.20±13.82)μm,A组、B组、C组角膜厚度均高于D组(132.40±2.56)μm,差异均有统计学意义(t=7.75、10.52、12.73,均P<0.001);造模后第7天,A组、B组、C组角膜厚度依次为(216.00±21.12)、(222.80±11.12)及(230.80±11.31)μm,A组、B组、C组均显著高于D组,差异均有统计学意义(均P<0.001),A组显著低于C组,差异有统计学意义(P=0.010);造模后第14天,A组角膜厚度为(150.80±12.86)μm,显著低于C组(203.40±19.29)μm,差异有统计学意义(t=-2.27,P=0.036);A组与D组相比较差异无统计学意义(t=1.40,P=0.178)。HE染色显示:造模第14天,A组和B组角膜基质层CNV数量及炎性细胞浸润均较C组降低。免疫组化示:A组和B组角膜组织中VEGF、IL-1β、TNF-α蛋白表达均低于C组。此外,Elisa结果显示A组角膜组织中VEGF蛋白含量为(50.85±1.73)pg/mL,显著低于B组的(62.11±1.33)pg/mL和C组的(76.81±2.38)pg/mL,差异均有统计学意义(均P<0.001)。结论结膜下注射阿柏西普联合应用妥布霉素地塞米松滴眼液可抑制碱烧伤后大鼠角膜CNV,并能促进角膜水肿的吸收。

Objective To investigate the role of aflibercept combined with tobramycin dexamethasone in inhibiting corneal neovascularization(CNV)in rats after alkali burn.Methods Thirty Sprague Dawley(SD)rats of specific pathogen free(SPF)grade,aged 6 to 8 weeks old,were selected and were divided into four groups based on random number table methods,with 10 rats in each group.Among which,CNV models in group A,B and C were induced by corneal alkali burn in SD rats.The rats in group A were treated with 2 mg/0.05 mL aflibercept by subconjunctival injection on the first day after modeling combined with tobramycin dexamethasone eye drops 4 times/day for 14 days.The rats in group B were treated with tobramycin dexamethasone eye drops 4 times/day for 14 days.The rats in group C(model control group)did not receive any treatment.And 10 normal SD rats in group D did not take any treatment.The thickness of the cornea was observed and compared on the 3rd,7th and 14th day after modeling,and the ratio of CNV area to corneal area was calculated.On the 14th day after modeling,all rats were executed.HE staining and immunohistochemistry were used to detect the expression levels of vascular endothelial growth factor(VEGF),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in corneal tissues,and the protein concentration of VEGF was detected by enzyme-linked immunosorbent assay(Elisa).Results On the 3rd day after modeling,the percentage of CNV to total corneal area in group A[2.97%(2.46%,4.15%)]and group B[3.00%(1.97%,6.37%)]were significantly smaller than the 5.67%(4.25%,7.36%)in group C,and the differences were statistically significant(t=-2.72,-2.08;P=0.007,0.037).On the 7th and 14th days after modeling,the percentage of CNV to total corneal area in group A was 5.06%(3.28%,6.43%)and 4.95%(3.02%,7.00%),which were significantly smaller than the 7.83%(5.61%,9.65%)and 8.64%(6.48%,10.15%)in group B,and the 7.97%(6.42%,12.32%)and 10.15%(6.26%,15.31%)in group C,and the differences were statistically significant(all P<0.005).On the 3rd and 7th day after modeling,the corneal thickness was(302.00±21.73)μm and(216.00±21.12)μm in group A,(294.80±15.23)μm and(222.80±11.12)μm in group B,and(311.20±13.82)μm and(230.80±11.31)μm in group C,which were significantly higher than the(132.40±2.56)μm in group D,and the differences were statistically significant(t=-7.75,10.52,12.73;all P<0.001).On the 14th day after modeling,the corneal thickness in group A was(150.80±12.86)μm,which were significantly lower than the(203.40±19.29)μm in group C,and the difference was statistically significant(t=-2.27,P=0.036).Besides,the difference of corneal thickness in group D was not statistically significant when compared with that in group A(P=0.095).On the 14th day after modeling,HE staining of corneal sections showed that scattered inflammatory cell infiltration and neovascularization in the corneal stroma in group A and group B were lower than those in group C.Immunohistochemistry showed the expression of VEGF,IL-1βand TNF-αprotein in corneal tissues in group A and group B were lower than those in group C.In addition,the expression of VEGF protein in corneal tissues in group A was(50.85±1.73)pg/mL,which were significantly lower than the(62.11±1.33)pg/mL in group B and the(76.81±2.38)pg/mL in group C,the differences were statistically significant(all P<0.001).Conclusion Subconjunctival infection of aflibercept combined with tobramycin dexamethasone eye drops can inhibit CNV in rats after alkali burn and promotes the absorption of corneal edema.