Research On The Effect And Molecular Mechanism Of Inflammatory Cytokines To The Blood-retinal Barrier Breakdown Induced By Anterior Segment Intraocular Surgery

Part 1 Rat model of blood-retinal barrier breakdown induced by anterior segment intraocular surgery.Objective:To establishes a practical rat model of BRB breakdown induced by anterior segment intraocular surgery.Methods:A 27-gauge needle attached to infusion tubing running to a bottle of balanced salt solution was inserted through the transparent cornea before the limbus into the rat anterior chamber.The pressure of the balanced salt solution was oscillated from 0 to 12 mmHg above the atmospheric pressure for 60 times.The needle was removed and the anterior chamber was formed.The contralateral eye was left untreated.Eyes were applied with ofloxacin ophthalmic solution after surgery.At 1^st,2^nd,3^rd,5^thand 7th after surgery, the integrity of the BRB was assessed by fundus photograph,fluorescence fundus angiography,optical coherence tomography,immunohistochemical staining for serum albumin or quantitative measurement using Evan's blue as a tracer.Results:Examination of fundus showed that most appeared normal.The fluorescein angiogram showed enlarged vascular diameter and enhanced background fluorescence intensity(partly visible vascular sheath in the late phase).1st day after surgery,the model group showed a statistically significant elevation in the central retinal thickness and the retinal Evans blue leakage as compared to contralateral control group and the normal control group(OCT:F=20.288,P＜0.001;Retinal Evans blue leakage:F=8.227,P=0.002). These increased and reached the peak at 2nd day after modeling and decreased to the point that there was no significant difference as compared to the contralateral control group and the normal control group at 5th day after modeling(OCT:d2 F=42.756,P＜0.001;d3 F=28.244,P＜0.001;d5 F=2.945,P=0.070;d7 F=0.699,P=0.506.Retinal Evans blue leakage:d2 F=33.360,P＜0.001;d3:F=41.305,P＜0.001;d5:F=0.091,P=0.913;d7: F=0.389,P=0.681).1st day after surgery,the model eyes showed that the albumin was abundant around the retinal vasculature in the nerve fiber layer.The extensive leakage of albumin was in the outer nuclear layer and the outer plexiform layers.2nd days after surgery,the albumin increased diffusely throughout the retina.At five days after surgery the albumin decreased revealing focal areas of staining adjacent to retinal vessels.There was also mild diffuse staining in the outer retina.At seven days after surgery the albumin was within retinal vessels.But there was little or no staining for albumin within the parenchyma of the retina.Inflammation was not evident in the retina.The extravasation of fluorescein,the increased central retinal thickness,strong staining for albumin and substantially elevated retinal Evans blue leakage demonstrated BRB breakdown after anterior segment intraocular surgery.Conclusions:This study establishes a practical rat model of BRB breakdown induced by anterior segment intraocular surgery.Part 2 Role of inflammatory cytokines in the blood-retinal barrier breakdown induced by anterior segment intraocular surgeryObjective:To clarify the role of inflammatory cytokines(prostaglandin E1,prostaglandin E2,prostaglandin F2α,interleukin-1βand tumor necrosis factor-α)in the blood-retinal barrier breakdown induced by anterior segment intraocular surgery.Methods:1.Detection of the inflammatory cytokines concentrations in the aqueous humor and vitreous of the rat model:rats were divided randomly into the control group and the model group.At 4h,1d,2d,3d,5d and 7 d after model,the concentrations of PGE1,PGE2,PGF2α,IL-1βand TNF-αin the aqueous humor and vitreous of the rat model were detected by enzyme-linked immunosorbent assay technique(Elisa technology).2.Effect of inflammatory cytokines injected into the anterior chamber to the blood-retinal barrier:rats were divided randomly into the blank control group,the negative control group and the cytokine group.The blank control group left untreated.The negative control group was injected 10 uL physiological salt solution into the anterior chamber by micro syringe.The cytokine group was injected 10 uL cytokine(PGE1,PGE2,PGF2α, IL-1βand TNF-α,respectively)into the anterior chamber by micro syringe.4h and 48h after injection,the concentrations of PGE1,PGE2,PGF2α,IL-1βand TNF-αin the aqueous humor and vitreous of the rat model were detected by Elisa technology.The integrity of the BRB was quantitatively measured using Evan's blue as a tracer.3.Effect of inflammatory cytokines antagonist to the blood-retinal barrier breakdown induced by inflammatory cytokines:for PGE1,PGE2 and PGF2αexperiment,rats were divided randomly into the negative control group,the model group,the NSAIDs prophylactic treatment group,the NSAIDs treatment group,the corticosteroid prophylactic treatment group and the corticosteroid treatment group.The negative control group was administrated with physiological salt solution eyedrop.The model group was modeled and administrated with physiological salt solution eyedrop.The NSAIDs prophylactic treatment group was modeled and administrated with Pranopulin eyedrop 2 days before modeling.The NSAIDs treatment group was modeled and administrated with Pranopulin eyedrop soon after modeling.The corticosteroid prophylactic treatment group was modeled and administrated with Prednisolone Acetate eyedrop 2 days before modeling. The corticosteroid treatment group was modeled and administrated with Prednisolone Acetate eyedrop soon after modeling.For IL-1βand TNF-αexperiment,rats were divided randomly into the negative control group,the model group and the treatment group.The negative control group was injected 10 uL the physiological salt solution into the anterior chamber by micro syringe after modeling.The model group was modeled and injected 10 uL the physiological salt solution into the anterior chamber by micro syringe after modeling.The treatment group was modeled and injected 10 uL cytokine antagonist into the anterior chamber by micro syringe after modeling.4h and 48h after injection,the concentrations of PGE1,PGE2,PGF2α,IL-1βand TNF-αin the aqueous humor and vitreous of the rat model were detected by Elisa technology.The integrity of the BRB was quantitatively measured using Evan's blue as a tracer.Results:1.Detection of the inflammatory cytokines concentrations in the aqueous humor and vitreous of the rat model:Elisa showed that 4h after modeling,the model group showed a statistically significant elevation in the concentrations of PGE1,PGE2 and PGF2αin the aqueous humor and retina.These increased and reached the peak at 1st day after modeling and decreased to the point that there was no significant difference as compared to the normal control group at 5th day after modeling.4h after modeling,the model group showed a slightly elevation in the concentrations of IL-1βand TNF-αin the aqueous humor and retina.These increased significantly at 1^stday and reached the peak at 2^ndday after modeling and decreased to the point that there was no significant difference as compared to the normal control group at 7th day after modeling.2.Effect of inflammatory cytokines injected into the anterior chamber to the blood-retinal barrier:4h after injection,Elisa showed that an elevation in the concentrations of PGE1,PGE2,PGF2α,IL-1βand TNF-αin the aqueous humor and vitreous in the negative control group compared to the blank control group.Both groups were significantly lower than those of the cytokine group.48h after injection,Elisa showed that the concentrations of PGE1,PGE2 and PGF2αin the aqueous humor and vitreous in the negative control group were similar to those of the blank control group.Both groups were significantly lower than those in the cytokine group.The retinal Evans blue leakage of three groups were similar at 4h after injection.The retinal Evans blue leakage in the negative control group was similar to those of the blank control group at 48h after injection.Both groups were significantly lower than those of the cytokine group.3.Effect of inflammatory cytokines antagonist to the blood-retinal barrier breakdown induced by inflammatory cytokines:4h after modeling,Elisa showed that the concentrations of PGE1,PGE2 and PGF2αin the aqueous humor and vitreous in the negative control group were similar to those of the NSAIDs prophylactic treatment group. Both groups were significantly lower than those in the model group.The concentrations of PGE1,PGE2 and PGF2αin the aqueous humor and vitreous in the corticosteroid prophylactic treatment group were higher than those in the negative control group,but lower than those in the model group.The concentrations of PGE1,PGE2 and PGF2αin the aqueous humor and vitreous in the NSAIDs treatment group and the corticosteroid treatment group were similar to those in the model group,higher than those in the negative group.The differences among the six groups were statistically significant.48h after modeling,The concentrations of PGE1,PGE2 and PGF2αin the aqueous humor and vitreous in the NSAIDs treatment group and the corticosteroid treatment group were lower than those in the model group,but still higher than those in the negative group.The differences among the six groups were statistically significant.The retinal Evans blue leakage in six groups was the same at 4 h after modeling.The retinal Evans blue leakage in the NSAIDs prophylactic treatment group was the least in the six groups at 48h after modeling.The differences among the six groups were statistically significant.The concentrations of IL-1βand TNF-αamong the negative control group,the model group and the treatment group were not statistically significant at 4 h after modeling.The concentrations of IL-1βand TNF-αin the negative control group were similar to those in the treatment group,both were lower than those in the modeling at 48 h after modeling. The differences were statistically significant.The retinal Evans blue leakage in three groups was the same at 4 h after modeling.The retinal Evans blue leakage in the treatment group was lower than that in the modeling,but higher than the negative control group at 48h after modeling.The differences among the three groups were statistically significant.Conclusions:Inflammatory cytokines(prostaglandin E1,prostaglandin E2,prostaglandin F2α,interleukin-1βand tumor necrosis factor-α)play an important role in the blood-retinal barrier breakdown induced by anterior segment intraocular surgery. Part 3 Effect and molecular mechanism of inflammatory cytokines on the barrier function of cultured human retinal pigment epithelial cell and human retinal vascular endothelial cellObjective:To clarify the molecular mechanism of inflammatory cytokines(prostaglandin E1,prostaglandin E2,prostaglandin F2α,interleukin-1βand tumor necrosis factor-α) induced the blood-retinal barrier breakdown.Methods:1.Human retinal pigment epithelial cell(HRPE)and human retinal vascular endothelial cell(HREC)were cultured on microporous filter-supports(Transwell cell culture plate)to establish a monolayer cell barrier.2.Effect of inflammatory cytokines to the barrier function of the HRPE cell and the HREC cell:cells were divided into the blank control group,the negative control group and the cytokine group.The blank control group did not seed the cells in order to detect the background.The negative control group was seeded HRPE cells or HREC cells and cultured with the medium without cytokines.The cytokine group was seeded HRPE cells or HREC cells and cultured with the medium with cytokines(PGE1,PGE2,PGF2α,IL-1βand TNF-α,respectively).48h after co-cultured,the transepithelial electrical resistance (TER)of HRPE cellar barrier or HREC cellar barrier were measured using an STX-2 epithelial voltmeter.The apparent permeability coefficients(Papp)of FD-70 of HRPE cellar barrier or HREC cellar barrier were measured using a fluorescence spectrofluorometer.3.Molecular mechanism of inflammatory cytokines to the barrier function of the HRPE cell and the HREC cell:cells were divided into the negative control group and the cytokine group.The negative control group was seeded HRPE cells or HREC cells and cultured with the medium without cytokines.The cytokine group was seeded HRPE cells or HREC cells and cultured with the medium with cytokines(PGE1,PGE2,PGF2α,IL-1β and TNF-α,respectively).48h after co-cultured,the expression of ZO-1,occludin and claudin-1 on the HRPE cells or HREC cells were labeled by immunofluorescence technique and observed using an laser scanning Confocal Microscopy.The expression of ZO-1,occludin and claudin-1 mRNA in the HRPE cells or HREC cells were detected by a reverse transcriptase polymerase chain reaction(RT-PCR).The expression of ZO-1, occludin and claudin-1 on the HRPE cells or HREC cells were semiquantitatively analyzed by Western blot.Results:1.Effect of inflammatory cytokines injected into the anterior chamber to the blood-retinal barrier:TER of normal HRPE cell barrier was 54.05±4.65Ω·cm2 and that of normal HREC cell barrier was 84.12±6.64Ω·cm2.A significantly decrease of TER occurred in cytokine group(PGE1,PGE2,PGF2α,IL-1βand TNF-α-supplemented medium)than in the negative control group after 48h co-cultured.The apparent permeability coefficient(Papp)of FD-70 of normal HRPE cellar barrier was 0.280±0.088×10^-3cm/s and that of HREC cellar barrier was 0.236±0.059×10^-3cm/s.A significantly greater increase of FD-70 sodium fluorescein occurred in cytokine group(PGE1,PGE2, PGF2α,IL-1βand TNF-α-supplemented medium)than in the negative control group.2.Molecular mechanism of inflammatory cytokines to the barrier function of the HRPE cell and the HREC cell2.1 Immunofluorescence labeled by-laser scanning Confocal Microscopy showed that ZO-1,occludin and claudin-1 mainly located at the cell junction.The distribution of ZO-1, occludin and claudin-1 in normal control HRPE cell or HREC cell were uniformity and continuous,while those in cytokines treated HRPE cell or HREC cell were nonuniformity and intermittently.2.2 RT-PCR showed that the expression of the ZO-1,occludin and claudin-1mRNA was observed.The expression of ZO-1,occludin and claudin-1mRNA was downregulated after 48h co-culture with PGE1,PGE2,PGF2αand TNF-α.The differences between the cytokine group and control group were statistical significance.The expression of occludin mRNA was downregulated and that of claudin-1 mRNA was upregulated after 48h co-culture with IL-1β.The differences between the cytokine group and control group were statistical significance. 2.3 Western Blotting showed that the expression of the ZO-1,occludin and claudin-1 was observed.The expression of ZO-1,occludin and claudin-1 was downregulated after 48h co-culture with PGE1,PGE2,PGF2αand TNF-α.The differences between the cytokine group and control group were statistical significance.The expression of occludin was downregulated and that of claudin-1 was upregulated after 48h co-culture with IL-1β.The differences between the cytokine group and control group were statistical significance.Conclusions:PGE1,PGE2 and PGF2αinduced blood-retinal barrier breakdown by slightly downregulating the expression of ZO-1,occludin and claudin-1 on the HRPE cell and HREC cell at the mRNA and protein level,resulting in destroying the tight junction on the HRPE cell and HREC cell.IL-1βinduced blood-retinal barrier breakdown by significantly downregulating the expression of occludin on the HRPE cell and HREC cell at the mRNA and protein level,resulting in the disbalance of occludin and claudin-1 and destroying the tight junction on the HRPE cell and HREC cell.TNF-αinduced blood-retinal barrier breakdown by downregulating the expression of ZO-1,occludin and claudin-1 on the HRPE cell and HREC cell at the mRNA and protein level,resulting in destroying the tight junction on the HRPE cell and HREC cell.