| Purpose: The characteristics of corneal structure without vascularization make it tobecome important structure of the visual organ in keeping completely transparent. Ininflammation, trauma, infection, hypoxia and other conditions, vascular network of corneallimbal will proliferate to form corneal neovascularization(CNV). CNV can make thephenomenon of corneal anterior segment simmuneprivileged disappearred, persistentinflammation of the cornea, corneal scar tissue, missing of the transparency of cornea, andsevere cases can cause blindness. How to prevent decreased vision caused by CNV is theimportant aspect of corneal basic research. In the research on tumor angiogenesis, aimingat the Dll4(important ligands of Notch1/Dll4signaling pathways)inhibitory treatmentachieved good results, but the signal pathways in the role and mechanism of CNVgenerated is unclear. This study intends to preliminarily discuss the role of Notch1/Dll4signaling pathways in the CNV, expecting to provide new targets for the treatment of CNVand theoretical basis.Methods:(1) Collected corneal neovascularization tissue samples of26cases througheyeball removal or corneal transplant surgery caused by all sorts of reasons, usingimmunohistochemical method and Rt-pcr test the amount of protein and mRNA expressionof Notch1and Dll4so as to clear receptor and the ligand of signal pathway expressed inmature newborn blood vessels.(2) To establish new blood vessels model through ratcorneal alkali burn, right eye for experimental eyes, left eye for black control eye. Inmodeling after1day,3day,5days, Dll4antibody of Notch1/Dll4signaling pathwaysspecific inhibitors and saline was injected subconjunctival in right eye respectively.7days,10days,14days after modeling using immunohistochemical method and Rt-pcr test theamount of protein and mRNA expression of Notch1and Dll4. At the same time, using themethod of CD31detect microvascular density of cornea. To clear protein and mRNAexpression trend of Notch1and Dll4in early stage of corneal neovascularization formationand the status of the growth of corneal neovascularization.(3) Methods in the second partis adopted to establish the alkali burn animal models and injected subconjunctival in righteye respectively. corneal confocal microscope was adaped to dynamically observe thechanges of the ultrastructure of rat cornea, including corneal epithelial cells, cornealstroma layer vascular perfusion, counting stromal fibroblasts, the number of inflammatorycells and form change at1,3,5,7,10,14days after modeling.At the same time, the general form of the corneal changes, appear time of new blood vessels, the area of cornealneovascularization, corneal turbidity in Rats were observed through the slit lampmicroscope.Results:(1) Expression amount of DLL4and Notchl in mature cornealneovascularization is low confirmed by immunohistochemical SP method and rt-pcrmethod, but the both express the synthetic change trend.(2) In the line of DLL4antibodytest in rat corneal alkali burn:7days after the modeling, the relative expression quantity ofDll4mRNA was the minimum in experimental group compared with control group byrt-pcr method, then gradually rise, to14days to express quantity highest; multiplecomparison,7days and10days after modeling the relative expression quantity has nostatistical significance (P>0.05);7days and14days,10days and14days after modelingthe relative expression quantity have statistical significance (P <0.00).7days aftermodeling the relative expression quantity of Notch1mRNA is minimum in experimentalgroup compared to control group, then gradually rise, to14days to express quantityhighest; Multiple comparison,7days and14days, seven days and10days,10days and14days after modeling the relative expression quantity of Notch1mRNA had statisticalsignificance (P <0.00). The relative expression quantity of Notch1mRNA and Dll4mRNAhad synthetic expression trend. Immunohistochemical resulets shows consistent trends inprotein expression and mRNA expression at each time point.7days after modeling,expression amount of Dll4protein and Notch1protein is the high level,10days aftermodeling to continue higher,14days after modeling gradually reduce in the control group;however, expression amount of Dll4protein and Notch1protein is the low level7daysafter modeling, then increased10days after modeling and14days after modeling is notobvious, there is shows stable low expression quantity in the experimental group. Theresults of half a quantitative analysis suggest protein expression quantity line from thestudy group was obviously lower than the control group at7days,10days after themodeling, difference has statistical significance (P <0.05);, there was no statisticallysignificant difference in experimental group and control group at14d after the modeling (P>0.05). New blood vessels density observation by CD31in study group and control group:7days after the modeling, new blood vessels density is (21.40+1.90)/HP in theexperimental group, the control group is (20.14+1.92)/HP, there is no statisticaldifference between two groups (P>0.05).10days,14days after the modeling, thenewborn blood vessels density is sparse in the experimental group compared with control group, there are statistically significant (P <0.05).(3) Test results of third part indicate: theaverage appear time of the corneal neovascularization in study group was (3.4+0.5) d, andthe control group was (3.2+0.6) d, there is no statistical difference between two groups (P>0.05). The largest area of new blood vessels in control group was (39.19+1.08) mm2,the experimental group was (17.43+0.64) mm2, corneal neovascularization area at eachtime point between two groups have statistical significance (p <0.01).Cloudy, edemadevelopment to peak at7days after the modeling in experimental group, the control groupto peak at10days after the modeling;7,10,14days after the modeling postoperatively, theexperimental group corneal turbidity is low than those compared with the control group,there was statistically significant difference (P＜0.05). Corneal confocal microscoperesults shows:(1) change of the corneal epithelial: normal structure of corneal epithelial isdisappeared after modeling in the experimental group and the control group.theexperimental group is seen epithelial cell structure obscurly at7days after modeling, andthe normal structure can be found at14days after modeling. corneal epithelium contourpartially visible at14days after modeling in Control group, but its formation not very rules.(2) changes in the structure of corneal stromal layer: normal structure of corneal stromallayer is disappeared after modeling in the experimental group and the control group.neovascularization and inflammatory cell infiltration is visible at3days after modeling inthe control group. neovascularization and inflammatory cell infiltration is visible at5daysafter modeling in the study group.10days after modeling, fibroblasts is found in thecontrol group, but the experimental group were less number of fibroblasts.(3) perfusionstatus of limbal vascular network: vascular network of corneal limbal is expansion and apoint to the central cornea bulky new blood vessels in the control group, the experimentalgroup show the blood flow speed but blood vessels are not obvious expansion and nobulky neovascularization. At the same time, the experimental group appears only a smallamount of inflammatory cell infiltrates in the peak stage of alkali burn repair, while thecontrol group has higher inflammatory cells infiltration in the whole process of cornealinjury.(4) comparison of inflammatory cells of corneal stromal layer, fibroblastscounting after alkali burn: inflammatory cells were lower in experimental group than incontrol group at3days,10days after modeling, there is the difference between two groups(P <0.01)., there is no statistical difference at inflammatory cells between both groups at14days after modeling (P>0.05)., fibroblast number difference between theexperimental group and the control group was statistically significant at7d,10d,14d after modeling (P <0.01).Conclusion:(1)In the mature period corneal neovascularization, the protein andmRNA of Notch1and Dll4have low expression, this results shows control ability islimited according to the inhibition of signaling pathway in corneal neovascularization late.(2) The results of second part of experiment suggest Dll4antibody aimed at Notch1/Dll4signaling pathway inhibitor in start-up of corneal neovascularization will be able toeffectively restrain the regulatory role of signaling pathways, reducing the rate of bulkynew blood vessels formation, it can help prevent serious complications late.(3)Observation by means of the cornea in form and using confocal microscope microscopic,the corneal neovascularization start-up to signaling pathway inhibitor of Dll4antibodytherapy helps to corneal wound healing, reduce inflammation of the cornea, preventcorneal tissue fibrosis, a degree of speed up the limbal vascular blood flow velocity andmay maintain vascular perfusion. |
