| Objective : To establish mouse corneal sutures, imbedding basic fibroblast growth factor(bFGF) in anterior chamber and corneal allograft transplantation models, detect the growth condation of corneal neovascularization and new lymphatic in different animal models, detect the changes of the blood vessels and the factors relevant to lympgatic vessels in corpus ciliare choroideae.Methods : First, established mouse corneal neovascularization models by different methods . 1. the mouse corneal suturing model: selected 32 BALB/c mice, after anesthesia by intraperitoneal injection of ketamine and chlorpromazine, at first,corneal indentations were made in mice corneal centers by corneal ring with 2mm in diameter lightly pressing ,then, sutured cornea with 11-0 nylon thread by the vertical direction of indentation . 2. the imbedding basic fibroblast growth factor(bFGF) in anterior chamber model: 22 mice were randomly divided into two groups, A and B groups ,imbeded empty vehicle in group A anterior chamber , imbeded 80ng bFGF in group B anterior chamber. After the success of anesthesia, lamellar corneal incision was made in peripheral cornea with a sticker , and penetrate the anterior chamber,then ,the release drug containing bFGF or empty vehicle was implanted in the inferior anterior chamber angle,lastly, corneal incision was sutured with 11-0 nylon seam . 3. the allogenic cornea transplant surgery model : penetrating keratoplasty was operated on the right eyes of 22 BALB/c mice, donor's cornea from C57BL/6 mice. Second, immunofluorescence and pathology of the cornea and iris of the mice in each group. On day 3,5,7,14 after surgery, 2-4 eyeballs were taken to produce frozen section,the corneal stretched preparation, Iris stretched preparation,. immunofluorescent stained for LYVE-1,CD31 and CD11b, observed the growth of corneal vessels,lymphatic vessels and iris vessels as well as the changes in factors relevant with lymphatic. 7 days after surgery, eyeball paraffin section was done, and stained by hematoxylineosin(HE)to make histopathology detection. Third, detected the expression of VEGFR-3,VEGF-A,VEGF-C in cornea and iris by molecular biology method. Extracted mRNA from sutured cornea on day 7,14 after surgery and normal cornea and iris. Detected the expression of the growth factors relevant with lymphatic and their receptors VEGFR-3,VEGF-A,VEGF-C by RT-PCR method. Four, observed the ultramicrostructure of corneal neovascularization and lymphatic tissue .3 mice were taken to observe observed the ultramicrostructure of corneal neovascularization and lymphatic vessels by ransmission electron microscope,which were operated on by corneal suturing and microencapsulation method induced neovascularization on day 5 and 14 after surgery.Results : 1,observed the growth of neovascularization in animal models by slit lamp (1) the suture group : on day 1 after surgery,corneal limbal vessels engorged. On day 3,new vessels invade into cornea with curve or loop. On day 5 ,new vessels which were vertical to corneal limbal extended intensively into cornea. on day 7, new vessels furtherly grew into suture. On day 7, dermaled sutures out, new vessels gradually regressed, on day 14 ,few blood vessel ghosts were remained. (2)the imbedding bFGF in anterior chamber group: on day 1 after surgery,cornea was edema,the amber hyaline delayed releasing vehicle was seen before the inferio riris.On day 3,cornea remained edema ,corneal stroma thickened,corneal neovascularization began to grew , iridal blood vessels dilated. .On day 5, many new vessels which were vertical to corneal limbal extended intensively into cornea. On day 7, new vessels furtherly grew . On day 14, blood vessels became thin and regressed, few blood vessel ghosts were remained, the delayed releasing vehicle in anterior chamber shrinked. (3) the allogenic cornea transplant surgery group: on day 3 after surgery,the corneal graft was lightly edema,the new vessels in corneoscleral limbus dilated,few new vessels extended to the implanting bed verge. On day 7,the corneal graft became edema and opacitas, new vessels grew into the implanting bed ,some extened to graft. new vessels fastly regressed after corneal sutures were dismantled. On day 14,the corneal graft remained hyaline, and 3-4 weeks began to reject.2,immunohistochemical examination. (1) the suture group: On day 3 after surgery,new vessels began to grow, and on day 7,grew prosperitily,then On day 14 regressed.By observing corneal stretched preparation,found that CD31+ vascular net distribute in Ansiform only in the limbus of cornea and few LYVE-1+ lympgatic vessels in the form of dilatate caecum appear in the limbus of cornea. on day 7, many arborescent new vessels which were vertical to corneal limbal extended intensively into cornea.,the new vessels ends was sharp. LYVE-1+ lympgatic vessels with blood vessels in flame form extended into the corneal center,and the end was caecum,the lumens was coarse,the morphous was irregular,the sporadic LYVE-1 weakly positive arborescent cells were found in corneal stroma. On day 14, blood vessels became thin obviously,and the ends coincided into vessels loop, lympgatic vessels also became thin and short,the end was blunt round,some lympgatic vessels disjucted from the limbus corneal lympgatic vessels,presented the microsoma tubiform construction in corneal stroma, sporadic LYVE-1 positive arborescent cells were found in periphery corneal stroma,they were CD11b positive cells.By examining iris stretched preparation,the radiational iris blood vessels were found in nomal iris;on day 7 after suture surgery, blood vessels density increased, lumens obviously thickened,the ntercellular space of the vascular endothelial cells became wide.In nomal iris,there were few LYVE-1 positive arborescent cells which mostly distributed in margo pupillaris iridis, however, on day 7 after corneal suture or microencapsulation surgery ,these cells obviously increased, diffusely distributed in corpus ciliare choroideae, in the middle and outside iris as well as ciliary body side,these cells,density was higher, double immunofluorescence staining discovered that these CD11b+ and F4/80+ cells be a part of macrophages derived from marrow. (2) the imbedding bFGF in anterior chamber group: on day 7 after surgery, in eyeball frozen section ,found that corrnea thicken obviously, CD31+ blood vessels express generously in the whole cornea, corneal lympgatic vessels grow in the superficial and middle stroma;iris hyperplasy and pachynsis,many blood vessels express, many LYVE-1 positive cells lie in the layer of irid- lens,these cells distribute on the pigmented epithelium of iris,extending to ciliary body.In iris stretched preparation,found many LYVE-1 positive cells,their morphouses were irregular,they were filled in the interspace around iris blood vessels,but no obvious tubiform constructions were found,no LYVE-1 positive lymph vessels were found. (3) the allogenic cornea transplant surgery group: on day 3 after PKP surgery,made stretched preparation,the ansiform and sentus pachy blood vessels and lympgatic vessels were found in perim. On day 7,we found much expression of blood vessels and lympgatic vessels,part of them had arrived to corneal graft. CD11b staining manifested the infiltration of two cells in corneal stroma,one was arborescent macrophage,the other was round inflammatory cell. LYVE-1 positive macrophages were found around lympgatic vessels. On day 14,the quantity and density of blood vessels decreased, lumens became thin.Lympgatic vessels regressed slower,the end became blunt round. CD11b+ cells decreased obviously. On day 3 after PKP surgery,in iris stretched preparation,we found that blood vessel thicken, density increase,and LYVE-1+ cells nearly vanish. CD11b staining manifested the infiltration of many round inflammatory cells on iris, few arborescent cells be found. On day 7,iris blood vessels became bulky and stiff. LYVE-1 positive cells increased , their morphouses were irregular,the small ellipse cells and arborescent cells were found. Round CD11b+ cells decreased, macrophages increased than before. On day 14,iris blood vessels became thin and sparse, LYVE-1 positive cells increased obviously,most of them were arborescent. Many CD11b+ morphouses were found uniformly distribute in iris.3,RT-PCR detected the changes of some corneal and iridal factors after suture surgery The resout showed the up-regulation of the VEGFR-3 expresson in cornea and iris on day 7 after suture surgery, and the change tendency of growth factor VEGF-A and VEGF-C at different times in cornea and iris consistent.4,Observed the ultramicrostructure of corneal neovascularization and lympgatic vessels Lymphangial vent was much bigger, vessel wall was irregular,which was composed of sequentes monolayer endothelial cells.Except peri-nucleus part ,the other parts kytoplasm was very thin and sequentes,had no window,no perithelial cells outside and no basal membrane or had little staccato basal membrane which was found in endothelial cells junction sometimes. There were 3 lymphangial endothelial cells conjunction types,that was end to end conjunction, overlapping conjunction and compact conjunction.Conclusion : the corneal suturing method , the imbedding basic fibroblast growth factor(bFGF) in anterior chamber method and the allogenic cornea transplant surgery method could be used to establish animal model for corneal neovascularization and lympgatic vessels.The mechanisms that corneal neovascularization and lympgatic vessels grow and regress were different. The quantity and distribution of the LYVE-1 positive macrophages changed in iris of different corneal neovascularization and lympgatic vessels animal models,and the iridal lympgatic vessels were not found. After corneal transplantation surgery , CD11b+ and LYVE-1 positive macrophages in iris migrated in early time,and then were supplied with cells in the other parts.
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