The Effect And Mechanism Of 0.1% Bromfenac Sodium On The Rat Corneal Neovascularization Model

ObjectiveCorneal neovascularization(CoNV)is a common pathologic change of different numerous ocular surface disorders.It is one of the reasons of visual loss and even blindness.At present,the drugs used to treat CoNV have certain effects,but can lead to a variety of adverse reactions.Therefore,searching for drugs which can effectively inhibit CoNV and reduce adverse reactions has attracted more and more clinical attention.In this study,0.1% Bromfenac Sodium Eye Drops were used to intervene CoNV model induced by alkali burn.And the clinical manifestations of cornea and anterior chamber,the progress of CoNV area,the changes of corneal histopathology,the degree of inflammatory reaction and the expression of CoNV related factor COX-2 and VEGF in corneal tissue were observed in each group at different time after modeling.To investigate whether 0.1% Bromfenac Sodium Eye Drops could inhibit CoNV in rats and its possible mechanism.Methods1.A model of CoNV was established with different duration of alkali burn of adult male SD rats.After modeling,corneal conditions were observed every day to find the best modeling method.2.The model of CoNV was established with alkali burn in the right eye of rats,and randomly divided into the model control group(A),PBS group(B),0.1% Bromfenac Sodium eye drops group(C),and 0.1% Fluorometholone eye drops group(D).The next day eye drops of PBS,0.1% Bromfenac Sodium and 0.1% Fluorometholone were respectively applied to the groups of B,C and D for twenty-one consecutive days.The normal control group(E)was not treated and fed normally.3.The anterior segment photos of the right eye(experimental eye)of each group were collected by slit-lamp microscopy before modeling and at 1,3,7,14,21,28 days after modeling.The clinical manifestations of cornea and anterior chamber were observed,the degree of corneal opacity and edema was scored and the complication rates were recorded.4.The ratio of CoNV area of rats in each modeling group on day 1,3,7,14,21 and 28 after modeling was calculated.5.At postoperative 7,14 and 28 days,three rats in each group were sacrificed by overdose anesthesia method,then the whole right eyeballs were taken and paraffin sections were made followed by hematoxylin-eosin staining.The histopathology changes of corneal tissue were observed under light microscope.6.Immunohistochemical staining was performed on the prepared paraffin sections of rat eyeballs.The expression of CD45 and VEGF-A in the corneal tissue of rats were observed under light microscope.7.At 7,14 and 28 days after modeling,total RNA was extracted from corneal tissues of each group and RT-PCR were used to detect the mRNA expression of COX-2 and VEGF in corneal tissues.8.On the 7th,14 th and 28 th days after modeling,total protein was extracted from corneal tissues of each group and ELISA were used to detect the protein expression of COX-2 and VEGF in corneal tissues.Results1.The rat model of CoNV was established successfully with the best burning time of 45 seconds.2.Clinical manifestations and complications: The degree of corneal opacity and edema in each group were aggravated within 7 days after modeling,and these symptoms gradually reduced after 7 days.CoNV in group A and group B grew into the central corneal,while CoNV only grew to the peripheral cornea in group C and D at 14 days after modeling.At 7,14,21 and 28 days after intervention,the degree of corneal opacity and edema and the ratio of CoNV area of group A and group B were significantly higher than those in group C and D(all at P<0.05).Within 28 days after modeling,no corneal perforation was observed in group A,and the rate of hyphema was 20%.The rates of corneal perforation in group B,C and D were 10%,10% and 30%,respectively,and the rates of hyphema were 20%,30% and 10%,respectively.3.Corneal histopathologic changes: On the 7th day after alkali burn,the number of corneal epithelial cell layers in each group were decreased and their arrangement was irregular.In group A and B,the normal morphology of the corneal epithelium and stromal layer was significantly changed,with edema thickening,fiber shrinkage,and a small amount of inflammatory cells infiltration.In group C and D,the corneal stromal layer was slightly thickened and edema was alleviated.On the 14 th and 28 th days after modeling,keratinization of corneal epithelium,stromal edema was gradually decreased in each group,and no inflammatory cell infiltration was observed.4.Expression of CD45,COX-2 and VEGF: Immunohistochemical staining showed that the expression of VEGF-A was positive in the cornea stromal layer of each group on day 7 after modeling.The staining intensity of group A and B was stronger than that of group C and D,and very few CD45 positive cells were observed in group A and B.VEGF-A staining was gradually weakened,and no inflammatory cell infiltration was observed in each group on the 14 th and 28 th days after modeling.On the 7th day after modeling,the corneal COX-2 and VEGF mRNA and protein expression in group A and B were significantly higher than group C,D and E(all at P<0.05).On the 14 th day after modeling,mRNA and protein expressions of COX-2 and VEGF in group A and B were still higher than those in group E(all at P<0.05),COX-2 protein expression in group A and D was higher than that in group C(all at P<0.05),and there was no significant difference between other molding groups(P>0.05).Conclusion0.1% Bromfenac Sodium Eye Drops can reduce the degree of opacity and edema,inhibit the formation of CoNV,reduce the corneal histopathological changes and the degree of scarring after alkali burns,so as to achieve the effect.The mechanism may be to reduce the expression of COX-2 and the degree of inflammatory reaction,and inhibit the expression of VEGF.But there are some complications after long-term application,which can provide reference for clinical application.