Effect of urokinase on the outward permeability of blood-retinal barrier following local injection in rats

Abstract

Background Retinal vascular recanalization is key to the treatment of retinal vascular occlusive disease.Studies confirmed that urokinase by intravitreal injection inhibits the expression of occludin protein at tight junction complexes among retinal capillary endothelial cells. Objective This study was to observe the effects of urokinase via eye local injection on the outward permeability of blood-retinal barrier by detecting the concentration of intravitreal Evans blue (EB). Methods Sixty healthy Sprague-Dawley (SD) rats were randomly assigned to four groups, and the right eyes of the rats were used as experimental eyes.Urokinase of 4 μl (350 U) and the equal volume of PBS (0.01 mol/L) was intravitreally injected separately in the intravitreal urokinase group and the intravitreal PBS group, and 10 μl urokinase (1 000U) and the equal volume of PBS was injected via retrobulbar tissue respectively as the retrobulbar urokinase group and the retrobulbar PBS group.Twenty-four hours after injection of drugs, 0.5% EB 4 μl was intravitreally injected.Four hours later, the rats were sacrificed and the right eyeballs were excised for the extraction and drying.EB was extracted from dried vitreous by formamide.Then, the concentration of EB in formamide was determined by a formamide extraction-ultraviolet spectrophotometry method to calculate the concentration of EB in vitreous.The use and care of experimental animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission (2011 version). Results The rat vitreous body showed the light blue color in intravitreal urokinase group and the retinal vessels were visible under the microscope, and that in the retrobulbar urokinase group presented blue color.However, in the intravitreal and retrobulbar PBS group, rat vitreous exhibited the deeper blue color and retinas were invisible.Absorbance of EB in formamide was 0.181±0.008, 0.450±0.017, 0.330±0.009 and 0.436±0.012 in the intravitreal urokinase group, intravitreal PBS group, retrobulbar urokinase group and retrobulbar PBS group, respectively.The intravitreal EB concentrations in the intravitreal urokinase group were (0.266±0.014)g/L, which was lower than (0.667±0.026)g/L, (0.496±0.015)g/L and (0.657±0.017)g/L of the intravitreal PBS group, retrobulbar urokinase group and retrobulbar PBS group, showing significant different among the four groups (F=100.406, P<0.01), and the intravitreal urokinase group showed the lowest value in comparison with other three groups (all at P<0.01). Conclusions Local application of urokinase around eye can augment the outward permeability of blood-retinal barrier in rats. Intravitreal assay of EB after intravitreal injection is a feasible approach to the determination of outward permeability of blood-retinal barrier. Key words: Urokinase; Intravitreal injection; Evans blue; Blood-retinal barrier; SD rats