| Background and ObjectiveAlkali injury of the eye is a serious blinding disease. Corneal neovascularization (CNV) after alkali burn is the major cause that influences the vision. The blindness cause by CNV becomes a prominent problem in our country. Multiple factors take part in the complex process of CNV formation. The exact mechanism of CNV is unknown. There isn't ideal medical treatment of CNV though there are many therapies. Investigate the mechanism and the antiangiogenic drug is extremely important.Recombinant human angiostatin is a kringle-containing fragment of plasminogen. The kringle1-3(K1-3) is a potent inhibitor of angiogenesis in vivo. The antiangiogenic mechanism of K1-3 is unknown. The previous study about AS was focus on the antiangiogenic effect of tumor angiogenesis and the inhibition of vascular endothelial cells. There are no publications of K1-3 eye drops on CNV and corneal inflammatory cells and the expression of IL-1αin rat after alkali burn. Leukocyte depletion rat models were established to investigate the effect of leukocyte on CNV induced by alkali burn. Rat CNV models were established to investigate the anti-angiogenenesis effect of K1-3 eye drops on CNV and the effect on the expression of IL-1αand VEGF.Materials and Methods1. 30 SD rats were burned on central cornea of left eye by 1mol/L NaOH for 40s to observe the change of cornea and the development of CNV after alkali burn. Five rats were killed randomly at 6th hour(6h),1,3,7,14,21day(d) after alkali burn. The corneas were taken for histopathological examinations.2. 105 SD rats were devided into five groups at ramdon, five for normal control (the rats were sacrificed and corneas were taken for control before the experiment). 100 rats for experiment (four groups, 25 rats each group) were burned on the central cornea of left eye by 1mol/L NaOH for 40s. saline to the control group , dexamethasone eye drops to dexamethasone group , K1-3 eye drops with different concentrations to group K1,K2. The eye drops were applied four times daily. Observe the cornea and CNV by ophthalmic surgical microscope, calculated the area of CNV on day 3,7,14,21. Then 5 rats of each group were killed randomly and the corneas were taken for histopathological,transmission electron microscop(eTEM)examinations,Immunohistochemistry and ELISA test.3. The corneas were stained with hematoxylin and eosine and leukocytes were counted by microscope. Observe the cornea ultrastructure of group K2 and saline group by TEM.4. Leukocyte depletion and CNV rat models were established to observe the occurrence and development of CNV after alkali burn. Compare the number of inflammatory cells and the area of CNV between leukocyte depletion group and control group, determine the effect of leukocyte in the development of CNV.5. Test the expression of VEGF and IL-1αof cornea at different time after alkali burn between four groups. The average optical density and microvessel density of cornea were analyzed.Result1. CNV in rats is successful induced by 1mol/L NaOH for 40s. On 3rd day after alkali burn, short vessels at the edge of the cornea can be seen, on 7th,14th day the vessels become very dense, the vessels growth slower after that. There were leukocytes in the cornea 6h after alkali burn, the leukocytes increased on 1st day and at the top of peak on 3rd day after alkali burn, after three days, the leukocytes decreased.2. Cornea ultrastructure: after alkali burn, the junction between the epithelial basement membrane and the stroma was loosed, the semidesmosomes decreased. The extracellular space was wider and the fibroblasts were larger than normal. Mitochondrions and rough endoplasmic reticulums were inceased in the control group. The damage of cornea in group K2 was lighter than that in control group.3. On 1st,3rd,7th,14th day after alkali burn, the number of leukocytes in group K1,K2,dexamethasone was less than that in control group(P<0.05). On 21th day, no significant difference was found between K1,K2 group and control group(P>0.05). But the leukocytes in dexamethasone group were increased. The MVD and area of CNV in group K1,K2 was smaller than that in group control(P<0.05).4. Significant difference(P<0.05)in the number of leukocytes,lymphocytes,neutrophils was found between cyclophosphamide group and control group. Area of CNV and the number of infiltrated inflammatory cells in the cyclophosphamide group were less than that in control group(P<0.05).5. VEGF was expressed in the epithelial weakly, the expression increased after alkali burn, the expression of VEGF arrived it's peak on 7th day. Significant difference(P<0.05)in the expression was found between K1-3 treated group and control group.6. Little IL-1αcan be tested in normal cornea, the expression of IL-1α increased after alkali burn and arrived it's peak on 3rd day , decreased on 7th day. Compared with control group, the expression of IL-1αdecreased significant(P<0.05)in K1-3 treated group.Conclusion1. Injury by 1mol/L NaOH for 40s is a simple and successful way to induced CNV. There is no severe complication take place in the cornea.2. The inflammatory cells play an important role in the occurrence and development of CNV, exhaust the leukocytes in the peripheral blood of rats can significant suppress inflammatory cell infiltration and the development of CNV.3. K1-3 can suppress inflammatory cell infiltration, inhibit inflammatory response, promote the healing of cornea and suppress the development of CNV in a dose-dependent manner.4. The anti CNV mechanism of K1-3 is that it can suppress inflammatory cell infiltration and inhibit the expression of VEGF and IL-1α.5. No toxicity was found in K1-3 treated cornea. K1-3 is hopeful to be applied in the future clinical therapy of CNV. |
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