| Part Ⅰ:Effects of Macrophages Infiltration on Suture Induced Corneal Neovascularization in RatObjective To investigate the effects of macrophages infiltration on suture induced corneal neovascularization(CNV)and corneal lymphangiogenesis(CL)in rat models.Methods The experimental animals were divided into three groups:Group1(blank liposome),Group2(macrophage scavenger),Group3(macrophage scavenger& macrophages).Rats in Group1 were injected subconjunctivally with 10ul blank liposome as control on day 1,4 and 8.Rats in Group2 were injected subconjunctivally with 10ul macrophage scavenger(Clodronate Liposomes)on day 1,4 and 8.Rats in Group3 were injected subconjunctivally with 10ul Clodronate Liposomes on day 1 and 4,and were injected subconjunctivally with 10ul macrophages(4×10~4)on day 8.Sutures were placed into the cornea stroma at a location of 1.5mm from the limbus on day 2.On day 7 and 14,1)Slit lamp was used to evaluate the degree of CNV and the severity of ocular surface inflammation;2)Hematoxylin-eosin(HE)stained corneas were observed under light microscope for evaluation of the changes of corneal structure in each group;3)Corneal immunofluorescence of CD68 and CD31 were used to evaluate the macrophages infiltration and the degree of CNV in each group.Results 1)Observation under the slit lamp:On day 7,the ratios of CNV area to the whole cornea of the three groups were 74.86±3.74%,57.00±2.85%and 64.00±3.20%,respectively.The ratio of CNV area of Group1 was higher than that of Group2 and Group3(Group2:P=0.0026,Group3:P=0.0318),and there was no statistical difference between Group1 and Group3(P=0.0674).On day 14,the ratios of CNV area in the three groups were 84.00±4.18%,62.51±3.12%and 81.31±4.06%,respectively.The ratios of CNV area of Group1 and Group3 were higher than that of Group2(Group1:P=0.0047,Group3:P=0.0035).2)HE staining:On day 7,the corneal stromal collagen of Group2and Group3 were more tightly arranged than that of Group1,and there were also less CNV and inflammatory cells infiltration in Group2 and Group3.On day 14,CNV and inflammatory cells infiltration of Group1 were still more than those of Group2.However,CNV and inflammatory cells infiltration of Group3 increased and became more than those of Group2.3)Immunofluorescence staining of CD68 and CD31:On day 7,the number of CD68-positive cells in each group were 12.83±4.40,1.33±1.40 and 103±0.86,respectively.The number of Group1 was higher than Group2 and Group3(Group2:P=0.0243,Group3:P=0.0204),and there was no statistical difference between Group2and Group3(P=0.8078).On day 14,the number of CD68-positive cells in each group were 9.69±2.39,1.87±1.12 and 14.46±4.86,respectively.The number of Group1 and Group3 were more than that of Group2(Group1:P=0.0021,Group3:P=0.0047). Immunofluorescence staining of CD31 showed a similar trend.On day 7,the CD31-positive region of Group1 was more than that of Group2 and Group3,and there was no significant difference between Group2 and Group3.On day 14,the CD31-positive areas of Group1 and Group3 were more than that of Group2.Conclusion Macrophages infiltration can promote the growth of CNV.Part Ⅱ:Effects and Mechanism of Corneal Collagen Cross-linking on CNV and CL Objective To investigate the effects and mechanism of corneal collagen cross-linking(CXL)on suture induced CNV and CL in rat models.Methods Rats were divided into three groups.CNV group:sutures were placed into the corneal stroma at a location of 1.5mm from the limbus to induce CNV and CL,and then the central corneal epithelia were scraped off;CXL group:rats in CXL group were performed CXL after sutures placed and central corneal epithelia scraped;CXL+ collagenase group:rats in CXL+collagenase group were treated with additional 5mg/ml collagenase for 30 minutes after the series procedures of sutures placed,epithelia scraped and CXL.On day 4,7,10 and 14,1)Slit lamp was used to evaluate the degree of CNV and the severity of ocular surface inflammation;2)HE stained corneas were observed under light microscope for evaluation of the changes of corneal structure in each group;3)Corneal immunofluorescence of CD68 and CD31 were used to evaluate the macrophages infiltration and the degree of CNV in each group;4)Real-time PCR was used to analyze the levels of m RNA expression of CD31,LYVE-1,CD68,i NOS and Arg-1,so as to study the growth of CNV and CL,the infiltration of macrophages and the polarization of M1 and M2 macrophages at m RNA level.Results 1)On day 4,the proportion of CNV area of CXL group and CXL+Collagenase group were higher than that of CNV group,though there was no significancant difference(CXL group:P=0.1292,CXL+collagenase group:P=0.1316);On day 7,10 and 14,the proportion of CNV area of CNV group and CXL+collagenase group were higher than that of CXL group(At the three time points,P values were 0.0341,0.0340 and 0.1004 for CNV group and 0.1648,0.0044 and 0.0188 for CXL+collagenase group,respectively).2)HE staining:On day 4,the corneal stromal collagen of CNV group was more tightly than that of CXL group and the CXL+Collagenase group,and there were also less CNV and inflammatory cells infiltration in CNV group.On day 7 and 10,CNV and inflammatory cells infiltration of CNV group and CXL+collagenase group were more than those of CXL group.On day 14,though CNV group had less inflammatory cells infiltration than before,CNV and inflammatory cells infiltration in CNV group and CXL+collagenase group were still higher than those in CXL group.3)Immunofluorescence staining of CD68:On day 4,the number of CD68-positive cells of CXL group and CXL+collagenase group were higher than that of CNV group,but there was no statistical difference(CXL group:P=0.0794,CXL+collagenase group:P=0.0971);On day 7 and 10,the counts in CNV group and CXL+Collagenase group were higher than that in CXL group(At the two time points,P values were 0.0100 and0.0064 for CNV group and 0.0010 and 0.0040 for CXL+collagenase group,respectively);On day 14,comparing with CXL group,the count of CD68-positive cells in CNV group was higher(P=0.0337);and it showed similar trend in CNV+Collagenase group,though there was no significant difference(P=0.1254).4)Immunofluorescence staining of CD31:On day 4,comparing with CNV group,there were more CNV in CXL group and CXL+collagenase group;On day 7 and 10,CNV in CNV group and CXL+collagenase group were more than that in CXL group;On day 14,CNV was still in the CNV group,but almostly disappared in CXL group and CXL+collagenase group.5)Real-time PCR:On day 4,the levels of relative m RNA expression of CD31,LYVE-1 and CD68 in CXL group were higher than those in CNV group.On day 7,10 and 14,although some comparisons were not statistically significant,the levels of relative m RNA expression of CD31,LYVE-1 and CD68 in CNV group and CXL+collagenase group were higher than those in CXL group.The ratio of relative m RNA expression of i NOS and Arg-1 in CXL group was higher than that of other two groups at day 4,7,10and 14.Conclusion CXL can inhibit the growth of CNV and CL by inhibiting the infiltration of macrophages,and increasing the stiffness of corneal stroma by CXL to promote the polarization of macrophages to M1 type,which may be one of the mechanisms of CXL inhibiting CNV and CL. |
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