| Objective: The mechanism of corneal neovascularization and the treatment has not been clarified yet, and drawed close attention recently. Vascular endothelial growth factor(VEGF) has been confirmed to play a key role in the formation of the corneal neovascularization. In recent study, hepatocyte growth factor(HGF) may also be an important regulator of angiogenesis, and discover that low-molecular-weight heparin (LMWH) has the effect to inhibit angiogenesis. But the relationship between HGF and CNV, and the inhibitory effect of LMWH on CNV are not reported recently. In order to observe the effect of LMWH on inhibiting corneal neovascularization after alkia burn and investigate its mechanism during this process, the expression of HGF, VEGF in cautery cornea was semi-quantitative analysed.Methods: Corneal alkali-burn model of 40 Wistar rats was made by burning right eyes with NaOH, and randomly divided into 2 groups : control group with normal saline subconjunctival injection, and experimental group with LMWH given by subconjunctival injection at 0.5IU/g body weight once a day for 7 days after the corneal alkaline burn. The other 8 Wistar rats is normal group without alkali burn. The rats were observed by slit lamp microscope after cautery and taken photoshops with digital camera. 5 rats were killed at 1,4,7 and 14 day randomly in each groups, the right cornea was extirpated, and the area of CNV were calculated. Half of every cornea were fixed in 4% paraformaldehyde, imbedded in paraffin to make tissue sections, stained and with HE for light microscope study. Immnuohistochemistry method. were used to investigate the distribution and expression of VEGF and HGF in rat cornea at different times. The rest of cornea were were fixed in low temperature ice box(-70℃)for relative quantitative reverse transcription polymerase chain reaction (RT-PCR) to observe the mRNA expression of HGF and VEGF. SPSS12.0 was used to analyse data of this study.Results: 1 Through lit lamp microscope: In control group, the vascular nets on limbus corneae engorged, the corneal epithelium amoticed on the 1st day after cautery; there were neovascularizations grew in limbus corneae fastly, the corneal epithelium repaired on the 2nd day; the neovascularizations encroached on corneal center on the 7th day; the neovascularizations became subsided though concentrate, and there were leukoma on the cautery region expressly on the 14th. In experimental group, the vascular nets on limbus corneae did not engorged, the corneal epithelium amoticed punctiformly on the 1st day; the neovascularizations grew in limbus corneae slowly, the neovascularizations were rarefaction than control group on the 4th,7th and14th day.2 Statistic result of area of CNV: On the 1st day after cautery, there were not new vessels in control group and experimental group. On the 4th, 7th and 14th day after cautery, the area (mm2) of CNV in control group and experimental group were respectively 15.10±0.18,28.82±0.37,39.45±0.45; 12.36±1.12,21.50±1.68,35.01±1.64. The area of CNV in experimental group were decreased significantly compared with control group in every time after cautery(t= 4.281, P<0.05 on the 4th day; t= 9.504, P<0.01 on the 7th day; t=5.808, P<0.01 on the 14th day).3 Immunohistochemistry analysis of HGF, VEGF.3.1 Statistic result of area density value of HGF: The cornea of normol rats expressed HGF, On the 1st,4th,7th and 14th day after cautery, the area density value of HGF in control group and experimental group were respectively 0.3199±0.0092, 0.6973±0.0102, 0.5071±0.0101, 0.3940±0.0540; 0.2950±0.0054, 0.4838±0.0102, 0.3713±0.0059, 0.3497±0.0137. On the 1st, 4th, 7th day after cautery, the area density value of HGF in experimental group were decreased significantly compared with control group.(P<0.05 on the 1st day, P<0.01 on the 4th, 7th day, P<0.05 on 14th day)3.2 Statistic result of area density value of VEGF: The cornea of normol rats did not expressed VEGF On the 1st,4th,7th and 14th day after cautery, the area density value of VEGF in control group and experimental group were respectively 0.3506±0.0077, 0.8746±0.0109, 0.5928±0.0910, 0.4477±0.0355; 0.3158±0.055, 0.6748±0.0960, 0.3970±0.0084, 0.3371±0.04208. On the 1st, 4th, 7th and 14th day after cautery, the area density value of VEGF in experimental group were decreased significantly compared with control group(both 1st and 4th P<0.05,both 7th and 14th P<0.01).4. RT-PCR analysis of HGF, VEGF4.1 The HGF mRNA content of rat cornea: The cornea of normol rats expressed HGF mRNA, On the 1st,4th,7th and 14th day after cautery, the expression of HGF mRNA in control group and experimental group were respectively 30.94±2.70, 78.08±3.06, 65.67±0.48, 28.62±4.90; 25.20±1.02, 49.25±4.60, 40.20±1.82, 23.05±4.80. On the 1st, 4th, 7th day after cautery, the expression of HGF mRNA in experimental group were decreased significantly compared with control group.(P<0.01 on the 1st, 4th, 7th, 14th day)4.2 The VEGF mRNA content of rat cornea:The cornea of normol rats did not expressed VEGF mRNA, On the 1st, 4th, 7th and 14th day after cautery, the expression of VEGF mRNA in control group and experimental group were respectively 40.46±3.26, 88.26±2.90, 68.80±5.40, 42.68±2.64; 31.20±1.92, 60.49±4.60, 54.68±1.80, 38.08±0.86. On the 1st,4th,7th day after cautery, the expression of VEGF mRNA in experimental group were decreased significantly compared with control group.(P<0.05 on the 1st, 14th day; P<0.01 on the 1st 4th, 7th day) Conclusion: 1 HGF and VEGF participate the process of corneal neovascularizantion, and play an important role in the corneal neovascularization.2 High expression of HGF and VEGF, and the broken balance in cornea leads the corneal neovascularization after cautery.3 LMWH suppresses the formation of corneal neovasculari- zation, possibly by inhibiting thrombus and fibrin that suppress the activation of vascular endothelial cell, then the expression of HGF and VEGF is depressed, moreover, endothelial cell migration and capillary tube formation is inhibitted.4 LMWH may become a new approach for the treatment of corneal neovascularization in future clinical work. |
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