Minocycline Inhibits Corneal Neovascularization By Regulating Macrophage Polarization After Alkali Burns In Rats

Objective: To investigate corneal neovascularization whether be inhibited by minocycline via regulating macrophage polarization after alkali burns in rats.Methods: A total of 72 healthy Sprague-Dawley(SD)rats of specific pathogen-free(SPF)grade,were randomly divided into three groups include control group(n=24,without treatment),solvent group(n=24,grade III alkali burns model in right eye)and minocycline group(n=24,grade III alkali burns model in right eye).After alkali burns,the solvent group was given intraperitoneal injection of 0.9% NaCL solution(10 mL/kg,qd)and minocycline group was given intraperitoneal injection of minocycline solution(45 mg/kg,bid).6 rats were randomly selected from each group on the 1st,4th,7th and 14 th day after alkali burns.The anterior segment of the eyes was observed by slit lamp,the length of corneal neovascularization and the growth area of corneal neovascularization were calculated at each time point.The inflammation of cornea was detected by Hematoxylin and Eosin staining,the mRNA relative expression of iNOS(marker of M1 macrophage)and Arg-1(marker of M2 macrophage)in corneal tissue was detected by Real time-PCR,and immunohistochemical staining was used to analyze the expression of TNF-?(marker of M1 macrophage)and CD206(marker of M2 macrophage)in corneal tissues.Results: 1.The results of slit-lamp examination showed that the length,number and area of corneal neovascularization in minocycline group were lower than solvent group on 4th,7th and 14 th days after alkali burns(P<0.01).2.The degree of corneal edema and the number of inflammatory cell infiltrations of minocycline group were lower than that of solvent group on 4th,7th and 14 th days after alkali burns(P<0.01).3.Compared to the control group,the solvent group was dominated by M1 macrophages(Arg-1/iNOS<1)on 4th and 7th days after alkali burns(P<0.01),macrophages were polarized towards M2 phenotype(Arg-1/iNOS>1)on 14 th day.Compared to the solvent group,The RNA expression of iNOS of minocycline group was decreased on 4th and 7th days after alkali burns,and the expression of Arg-1 was decreased on 14 th day(P<0.01).4.The results of correlation showed that the relative expression of Arg-1 was postively correlated to the length and area of corneal neovascularization in the solvent group(Pearson correlation coefficients were 0.898 and 0.781).5.Immunohistochemistry showed that the optical density values of TNF-? and CD206 in minocycline group were lower than those in solvent group on 7th and 14 th day after alkali burns(P < 0.05).Conclusion: 1.Macrophages promote the formation of new blood vessels after corneal alkali burn by polarizing to the M2 phenotype.2.Minocycline could reduce corneal neovascularization after alkali burns by regulating macrophage polarization.