| Objective:Corneal neovascularization(CNV) is a major challenge following cautery,which often leads to blindness.Recent studies have documented that VEGF,MMP-9 and NO were correlated with neovascularization in vivo. In order to evaluate the effect of VEGF,MMP-9 and NO in CNV, and the effect of L-NAME in the corneal epithelium healing,the content of NO was measured,and the changed expression of VEGF and MMP-9 in cautery cornea was qualitative and semi-quantitative analysed in Cautery group and L-NAME group.The ultrastructure changes of the corneal epithelium were observed at the same time.And this all will provide credible theory and aid in the design of new therapeutic approaches to treat and prevent CNV,and in the corneal epithelium healing after cautery. Methods:Corneal alkali-burn model of 40 New Zealand white rabbits was made. The rabbits were divided into Cautery group(cautery,n=20) and L-NAME group(cautery and L-NAME 10mg/kg/d ip,n=20) in random.The rabbits were observed by slit lamp microscope after cautery. 5 rabbits were killed at 1,4,7 and 14d randomly in each groups and the left cornea was extirpated.The content of NO were messured by enzymic method in half of every cornea.The quarter of every cornea were fixed in 4% paraformaldehyde, imbedded in paraffin to make tissue sections for light study and immunohistochemistry analysis.The computer image analysis system was used to analyse the expression of VEGF,MMP-9 in cornea.The rest of cornea were fixed in 4% glutaraldehyde-phosphate buffer quickly at the size of 1mm3 to make samples for transmission electron microscope for observing the ultrastructure changes of the corneal epithelium.SPSS12.0 was used to analyse data of this study.T-test and correlation analysis were used in the statistical management. Results: 1 Through lit lamp microscope,the results of corneal inflammation and neovascularization in L-NAME group were less than Cautery group. 2 Statistic result of area of CNV: On the 1th day after cautery, there were not new vessels in L-NAME group and Cautery group. On the 4th,7th and 14th day after cautery, the area(mm2) of CNV in Cautery group and L-NAME group were respectively20.93±0.65,29.73±1.78,48.72±2.62;16.70±0.64,21.87±0.97,34.74±1.47.The area of CNV in L-NAME group were decreased significantly compared with Cautery group in every time after cautery(both P<0.01). 3 Statistic result of content of NO: On the 1th,4th,7th and 14th day after cautery, the content(μmol/gprot) of NO in Cautery group and L-NAME group were respectively 6.58±0.07,6.90±0.03,6.41±0.03,6.04±0.05;6.44±0.06,6.61±0.05,6.21±0.02,5.95±0.03.On the 4th,7th day after cautery, the content of NO in L-NAME group were decreased significantly compared with Cautery group(both P<0.01). On the 1th,14th day after cautery, the content of NO in L-NAME group were lower than those in Cautery group(both P<0.05). At the same time, the area density value of VEGF ,MMP-9 all had significant positive correlation with the content of NO(r=0.962,p<0.01;r=0.704,p<0.01). 4 Immunohistochemistry analysis of VEGF,MMP-9 Immunolocalization of VEGF,MMP-9 in cornea were intracytoplasm of corneal epithelium cells,matrix fiber cells,infiltrative inflammatory cells and neovascular endothelium cells. 4.1 Statistic result of area density value of VEGF: On the 1th,4th,7th and 14th day after cautery, the area density value of VEGF in Cautery group and L-NAME group were respectively 0.0812±0.0025,0.1029±0.0044,0.0764±0.0023,0.0172±0.0008;0.0516±0.0026,0.0823±0.0019,0.0363±0.0015,0.0169±0.0005.On the 1th,4th,7th day after cautery, the area density value of VEGF in L-NAME group were decreased significantly compared with Cautery group(both P<0.01),but there were not significant difference between them (P>0.05) on the 14th day after cautery. 4.2 Statistic result of area density value of MMP-9: On the 1th,4th,7th and 14th day after cautery, the area density value of MMP-9 in Cautery group and L-NAME group were respectively 0.0447±0.0013,0.0305±0.0013,0.0175±0.0004,0.0149±0.0002;0.0345±0.0011,0.0245±0.0017,0.0164±0.0007,0.0149±0.0002.On the 1th,4th day after cautery, the area density value of VEGF in L-NAME group were decreased significantly compared with Cautery group(both P<0.01),and the area density value of VEGF in L-NAME group were lower than those in Cautery group(both P<0.05) on the 7th day after cautery, but there were not significant difference between them (P>0.05) on the 14th day after cautery. At the same time, there were positive correlation between the area density value of VEGF and those of MMP-9(r=0.555,p<0.01). 5 Histopathology change 5.1 Light microscope:Cautery group: The corneal epithelium healing was slowly; cell shape was irregularity; inflammatory cell infiltration was critical; corneal neovascularization was early and development was quick; substantia propria layel collagen fibers had segmentation and recovery was slowly. L-NAME group: The corneal epithelium healing was quick; cell shape was integrity; inflammatory cell infiltration was slight; corneal neovascularization was late,slow-moving and rarefaction; substantia propria layel collagen fibers arrange irregularity but its recovery was fast. 5.2 Electron microscope: Cautery group: corneal epithelium cell had a slow healing and less quantity; microvilli was rarefaction; the amount and functional recovery of cell organelle was slowly; epithelium cells connection form late. L-NAME group: corneal epithelium cell had a fast healing and more quantity; microvilli...
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