Empirical Study Of Urokinase Injected Into The Vitreous Cavity Can Penetrate The Retinal After Ligate The Optic Nerve In Rabbits

Background and ObjectiveCentral retinal artery occlusion(CRAO) is a serious disease which can lead the eyes to grow blind,it is retinal acute ischemia but we have not a ideal methods to treat it.Schmidt first applied urokinase for Selectivity ophthalmic artery thrombolysis in clinic in 1992,and it can relieve patients'retinal edema and improve retinal ischemia and eyesight.With the thrombolysis methods improve,some studys imply that local intervention thrombolysis can improve eyesight.but there are strict standards about case's internalize and reject to decide whether use local intervention thrombolysis to treat it,meanwhile it brings troubles to patient's treatment,evenmore there are some serious complications case reports such as hemiplegia. Those complications are resulted in Urokinase's widely effect, if we can find a method which can limit Urokinase's effect in eye, we can clearly avoid those central nerve system's serious complications.Urokinase(UK) is a most widely clinical application thrombolytic drug at present, there is not serious complication case reports when UK is used to anterior chamber flush,subconjunctival injection.UK is non-fibrin specificity plasminogen activator which have short half life and long time effectiveness as 12-24 hours for degrdn fibrin and blood coagulation factor,moreover it is no antigenicity for human body,it can not lead to anaphylactic response.Kamei find that intravitreal inject plasminogen activator can through retina into the subretinal space and lyse the subretinal clot in rabbits, and not find obvious side effects.But whether intravitreal inject plasminogen activator can treat the retinal vascular occlusion is not yet clear.We assumpt that there ia a possibility intravitreal inject UK can treat the retinal vascular occlusion, then we first need to observe if UK can penetrate the retinal vessel.This study injected urokinase into the uitreous cavity after ligate the optic nerve in rabbits to determine if urokinase injected into the vitreous cavity can penetrate the retinal vessels of rabbit eyes so that we can establish foundation of intravitreal inject UK treat the retinal vascular occlusion.Materials and Methods54 young rabbits were randomly divided into A, B and C 3 groups (18 in each group, respectively), A, B, and C. A,B groups as the experimental groups, C as the control group. Right eye of all animals were used as the study.Group A underwent temporary ligation of the optic nerve, and one eye of each rabbit was injected with the experimental groups were given 5000U/0.1ml of UK, one eye of each rabbit of group B was injected 5000U/0.1ml of UK without ligation of the optic nerve. An equivalent volume of 0.01ml sterile phosphate buffered saline (PBS) was injected into group C as control.Ten samples of right eyes in group A,B and two samples in group C were taken for pathological examinations and immunohistochemistry (IHC) study with the paraffin-embedded sections by scarifying the rabbits at 24 hours after intravitreal injection for each group.Image acquisition data can be obtained from Nikon DS-Ril biomicroscopy and compute the data of mean optical density (MOD) of each section with Image-Pro Plus 6.0 image analysis software.Statistical Treatment These datas of each section's Density Mean were analyzed by statistical software SPSS 17.0. Independent-Sample T Test was used to analyze the difference between the different groups.α=0.05 was considered as the level of tests.Results(1)The histomorphology of rabbit retina was observed with HE stained paraffin-embedded sections. the structure and morphology of retina in C group was prepared as standard control, no clearly histomorphology changing in group C was observed. However, cells reducing and wide nucleus gaps in INL and ONL was observed for both A and B groups and clear boundaries between retinal layer and smooth surface only in group B be found. edema of retinal surface and vague boundaries as well as appearing of irregular array of the structure of retina and vascular endothelial cell loose arrange in A group were revealed. Besides, some cells with wider nucleus gaps in INL and ONL can also be detected.(2) Immunohistochemistry analysis of the sample tissues has shown all negative in control (group C). Meanwhile, the control sections of experimental groups were also negative being compared to all other experimental groups that show positive expressions.Positive expression at inner limiting membrane(ILM),retinal nerve fiber layer (RNFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL),inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL)and cavity of the Retinal vessel could be found in group A. group B revealed positive expression at retinal nerve fiber layer (RNFL), retinal ganglion cell layer (RGCL), inner plexiform layer (IPL), and inner nuclear layer (INL).The structure and morphology of retina in C group was prepared as standard control, In comparison of the penetration for two groups A and B by Independent-Sample T Test at the same doses and the same time point, with or without ligation of the optic nerve, The differences between two groups had statistical significance (t=14.603, p<0.05).(3)Retinal toxicity of urokinase:no clearly histomorphology changing in group C was observed; meanwhile cells reducing,wide nucleus gaps in ONL, clear boundaries between retinal layer and smooth surface be found in group B.So we can know that the dose of 5000U/0.1ml UK have retinal toxicity.Conclusions(1) Urokinase injected into the vitreous cavity for 24 hours can penetrate the retinal vessel after ligation of the optic nerve in rabbits.(2) Urokinase has retinal toxicity.