| ObjectiveThe anti-VEGF medicine Conbercept,which is combined with vascular endothelial growth factor(VEGF)receptor,can make VEGF lose biological activity and inhibit angiogenesis and the proliferation of fibrous tissue.Study on the effect of different doses of post-treatment about Conbercept on the filtration bleb after trabeculectomy in rabbit eyes,by comparing with mitomycin C(MMC),to investigate whether it is possible to be a new candidate for anti scar formation after glaucoma filtering surgery.Methods1.The animal model of glaucoma surgery in rabbit eyes was established by the 30 healthy adult rabbits(n=60 eyes)of either gender with 2~2.5kg in weight treating with trabeculectomy after recording intraocular pressure(IOP).The 30 healthy adult rabbits(n=60 eyes)were randomly divided into 5 groups.Different dosages of drug were used in each rabbit's eyes and marked on the ears.(1)The first group: the right eye in 6 rabbits was injected with normal saline(0.1mL)under the operation,and the left one was treated with mitomycin 0.2g/L in the cotton piece that was placed under the scleral flap about 3 minutes,then washed with normal saline.(2)The second group: the left eye in 6 rabbits was injected with Conbercept(0.025mL)under the operation,and the other was treated the same as the first group with mitomycin.(3)The third group: the right eye in 6 rabbits was injected with Conbercept(0.025mL)under the operation,and the other was injected with Conbercept(0.05mL).(4)The fourth group: the right eye in 6 rabbits was injected with Conbercept(0.05mL)under the operation,and the other was injected with Conbercept(0.075mL).(5)The fifth group: the right eye in 6 rabbits was injected with Conbercept(0.075mL)under the operation,and the other was injected with normal saline(0.1mL).2.The filtration wound healing and conjunctival hyperemia about the surgical eye were observed at different time,including corneal edema,anterior chamber depth and inflammation,lens transparency.The morphology of filtering bleb about the surgical eye was observed and pictured.And intraocular pressure was measured at different time.The rabbits were killed after 7 days,15 days and 30 days,each group randomly selecting 2 rabbits,10 rabbits each time,completely removing postoperative eyeball,with 4% formalin fixed 24 h.The surgery regional organization on the rabbit eyes were embedded in paraffin for eosin staining,Vimentin staining and Sirius Red in Saturated Carbazotic Acid Staining.Eosin staining was used to observe and record the infiltration of inflammatory cells in the filtering channel by microscope(×200).The ? type(red or yellow double refraction)and ? type(blue green double refraction)collagen fibers were observed by polarized light microscope on the Sirius Red in Saturated Carbazotic Acid Staining.Five high power fields(×200)was randomly selected from each slice,and Image-pro plus7.0 software was used to analyze the proliferation of collagen fibers at different time after operation in rabbit eyes,taking the percentage of collagen fibers in the filtering area of the visual field as a standard.To observe the proliferation of the fibroblasts in the filtering tract with Vimentin staining,each slice randomly choosed 5 vision in operation area,counted positive cells(positive cells in the nucleus of the standard to judge brown yellow granulus')at high magnification(×200)for each field of view,the average value was taken as the number of positive cells in the slice.Results1.The anti-glaucoma surgery model in 60 eyes of rabbit was made by the same experienced doctor.Experimental results demonstrated that there was no significant difference between each group at the same time on changes of anterior segment,such as cornea,anterior chamber,lens,etc.After 7 days,there were different degrees of hyperemia in the conjunctiva of the operation area,and the filtering blebs were swelling and dispersing(functional follicle)in the experimental groups.And there was no statistically significant difference(P>0.05)between two groups.The follicles were gradually flattening and shrinking in the saline group after 7 days.After 30 days,the follicles in the area of the normal saline group were encapsulated,with obvious scar,thick wall and abundant blood vessels(nonfunctional follicle).The follicles were small and flat,the area was smaller in Conbercept(0.025mL)group,and most of them were functional follicle.The Conbercept(0.05 mL,0.075mL)groups and MMC group showed a thin wall,swelling,scattered like functional follicles.There was statistically significant difference(P<0.01)between saline group and Conbercept(0.025 mL,0.05 mL,0.075mL)groups after 30 days.After one day,the intraocular pressure in each group was significantly lower than that before operation,and the difference was statistically significant(P<0.01),but there was no significant difference between the two groups(P>0.05).The IOP increased gradually to the normal value from 15 days to 30 days after operation in the saline group,and there was no significant difference(P>0.05)before the operation about the IOP.After 15 days,the IOP increased gradually in the Conbercept(0.025mL)group,but it was lower than that before the operation.However,at the same time the IOP remained stable and low in Conbercept(0.05 mL,0.075mL)groups and MMC group.On the 30 days after surgery,the difference of IOP between Conbercept(0.05 mL,0.075mL)groups was statistically significant compared with Conbercept(0.025mL)group and the saline group(P<0.01).There was statistical significance of IOP in Conbercept(0.025mL)group and normal saline group(P<0.01),intraocular pressure difference was not statistically significant in Conbercept(0.05 mL,0.075mL)group and MMC group(P>0.05).2.Rabbits were killed in 7 days,15 days and 30 days after operation,completely removing postoperative eyeball,and the surgery regional organization on the rabbit eyes were embedded in paraffin for staining.(1)Eosin staining indicated that there were varying degrees of infiltrating inflammatory cell in the conjunctiva of each group,mainly eukomonocyte and plasma cells(eukomonocyte was small and round,karyoplasmic ratio,nuclear trachychromatic blue purple;the cytoplasm of the plasma cells slightly basophilic,deep and eccentric nuclear staining,plasma cell size between lymphocytes and macrophages / multinucleated giant cell).A large number of lymphocytes and neutrophil infiltration were observed in the normal saline group.But a few lymphocytes and infiltrating plasma cells can be observed in the conjunctiva,and the tissue structure was sparse in Conbercept(0.025 mL,0.05 mL,0.075mL)groups and MMC group.(2)The results of Vimentin staining suggested that the number of fibroblasts in Conbercept(0.05 mL,0.075mL)groups and the MMC group was less than that in Conbercept(0.025mL)group at the same time.And the number of fibroblasts in Conbercept(0.05 mL,0.075 m L)groups was nearly the same as that in the MMC group at the same time,there was no significant difference about fibroblast between the two groups in Conbercept(0.05 mL,0.075mL)group and MMC group at the same time(P>0.05).Compared with the Conbercept(0.05mL)group,the fibroblast of Conbercept(0.025mL)group and the normal saline group were significant difference at the same time(P<0.01).There was significant difference about fibroblast between the Conbercept(0.025mL)group and the normal saline group at the same time(P<0.01).(3)Sirius Red in Saturated Carbazotic Acid Staining showed that the content of type ? and type ? collagen fibers increased gradually in each group with the prolongation of time.The type ? and type ? collagen fibers in Conbercept(0.05 mL,0.075mL)group and MMC group at the same time were relatively sparse and regular,and they were not significantly different(P>0.05)from the three groups at the same time.Compared with the Conbercept(0.05mL)group,the type ? and type ? collagen fibers of Conbercept(0.025mL)group and the normal saline group were significant difference at the same time(P<0.01).The type ? and type ? collagen fibers were relatively dense and disordered in the saline group,and the type ? and type ? collagen fibers on the saline group were significantly different(P<0.01)from the Conbercept(0.025 mL,0.05 mL,0.075mL)groups.Conclusion1.Conbercept injecting in tunica conjunctiva after trabeculectomy was helpful to reduce the formation of the new blood vessels,inhibit the scar of the filter,and maintain the shape of the filtering bleb.2.There was no obvious side effect to the eye tissue on Conbercept.3.The effect of Conbercept(0.05 mL,0.075mL)dose on the anti-scar effect was stronger than that(0.025mL)dose.But there was no obvious difference on the anti-scar effect between 0.05 mL and 0.075 mL.4.Conbercept(0.05 mL,0.075mL)can effectively inhibit the proliferation of fibroblasts during the initial healing stage of rabbit eyes after glaucoma surgery so as to reduce the proliferation of fibroblasts and the synthesis of collagen type ? and ?.The experiment indicated that Conbercept can prevent filtering scar by inhibiting scar formation via anti-VEGF. |
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