| Purpose:In this study, corneal transplantation mice model with different genes wereestablished by surgical methods. Topical sphingosine1-phosphate receptormodulator1(S1P1) was applied to this model; clinical effects of S1P1on graftimmune rejection were observed after operation. Immunosuppressive agentscommonly used in ophthalmic such as dexamethasone (DXM), rapamycin (Rapa) andcyclosporine A (CsA) were compared with S1P1clinically. Mechanism of S1P1forinhibiting immunological corneal graft rejection was determined by detecting cornealdendritic cell-associated immune factors expression and protein levels; clinical effectsof topical S1P1combination were evaluated after operations clinically.Methods:1. Classical surgical method was use to establish corneal transplantation mice modelwith different genes. Corneal buttons of24Male C57BL/6mice (donor) weretransplanted to48male BALB/C mice (receptor) for the allogeneic transplantationexperimental models. One random eye in random mouse was selected as receptor,which was transplanted with male C57BL/6mouse corneal graft. All mice wererandomly divided into six groups (A, B, C, D, E and F groups), and each groupcontained8mice. Group A was set as control group which was treated with placeboeye drops without any drugs; group B was treated with0.25%S1P1suspension eyedrops; group C was treated with0.5%S1P1suspension eye drops; group D was treatedwith0.1%dexamethasone eye drops; group E was treated with0.4%rapamycin eyedrops; group F group was treated with1%cyclosporine A eye drops. Each group wastreated with topical different medicine for four times a day (8-12-16-20o’clock),continuous administration until the occurrence of corneal graft rejection. On10or11 day after operation, sutures were removed under a microscope. Mice were observeddaily postoperatively for corneal changes, corneal graft survival time was recorded.After corneal rejection, experimental animals were sacrificed with overdoseanesthesia.2. Classical surgical method was use to establish corneal transplantation mice modelwith different genes.30male BALB/C mice were randomly divided into five groups(G, H, I, J and K groups), and each group contained6mice. Group A was set ascontrol group which was treated with placebo eye drops without any drugs; group Hwas treated with0.5%S1P1suspension eye drops group, group I was treated with0.1%dexamethasone eye drops; group J was treated with0.4%rapamycin group;group K was treated with1%cyclosporine A eye drops. Each group was treated withtopical different medicine for four times a day (8-12-16-20o’clock). On10or11dayafter operation, sutures were removed under a microscope. On14d after surgery,experimental animals were sacrificed with overdose anesthesia. After that, unilateralcervical draining lymph nodes, peripheral blood, spleen were removed and tested withflow cytometry to detect the distribution and aggregation of CD11c+, MHC-II+andCD86+DCs. IL-2, IL-12, TGF-β1, IFN-γ and CTLA-4levels in blood serum weredetected with Elisa; three corneal buttons of each group were extracted. Total RNAwas extracted, and Realtime-PCR examination was performed to determine IL-2,IL-12, TGF-β1, IFN-γ and CTLA-4mRNA expression; three corneas in each groupwere fixed for routine H&E staining to evaluate corneal rejection conditions,immunohistochemical examination was performed to evaluate MHC-II+DCs andCD86+DCs distribution and infiltration in the cornea grafts.3．Classical surgical method was use to establish corneal transplantation mice modelwith different genes.32male BALB/C mice were randomly divided into four groups(L, M, N and O groups). Group L was set as control group and treated with drug-freeplacebo eye drops; group M was treated with0.1%dexamethasone eye drops; groupN was treated with0.5%S1P1suspension eye drops combined0.4%rapamycin eyedrops; group O was treated with0.5%S1P1suspension eye drops combined1%cyclosporine eye drops. Since the first day after surgery, mice were treated with topical ocular medicine four times a day (8-12-16-20o’clock); two ophthalmic eyedrops were given with interval of10minutes. Drugs were continuously applied untilthe occurrence of corneal graft rejection. On10or11day after operation, cornealsutures were removed. Mice were observed daily postoperatively for corneal changes,corneal graft survival time was recorded. After corneal rejection, experimentalanimals were sacrificed with overdose anesthesia.Results:1.48allogeneic keratoplasty grafts in mice were analyzed statistically. The cornealgraft mean survival time (MST) in control group was16.8±1.7days (n=8); the MSTin0.25%S1P1eye drops group was17.8±1.9days (p <0.01, n=8); the MST in0.5%S1P1eye drops group was27.6±3.3days (p<0.01, n=8); the MST in0.1%dexamethasone eye drops group was31.9±4.3days (p<0.01, n=8); the MST in0.4%rapamycin eye drops group was25.1±4.0days (p<0.01, n=8); the MST in1%cyclosporine A group was25.8±4.2days (p<0.01, n=8).2. Compared with the control group, MST in0.25%S1P1eye drops group had beenextended, but there was no significant difference (P0.05); MST in0.5%S1P1eyedrops group,0.1%dexamethasone eye drops group,0.4%rapamycin eye drops group,1%cyclosporine A eye drops group were all prolonged, and there were a significantdifferences in these groups (P<0.01, respectively). Compared with0.5%S1P1eyedrops group,0.1%dexamethasone group was significantly prolonged graft survivaltime, and there was significant difference (P <0.05). Compare with0.4%rapamycineye drops group and1%cyclosporine A eye drops group,0.5%S1P1eye drops groupprolonged MST, but there were no significant differences (P0.05, respectively).3.Topical application of0.5%S1P1eye drops significantly increased MHC-II+CD11c+cells ratio in peripheral blood (P<0.01), significantly reduced MHC-II+CD11c+cellsand MHC-II+CD86+ratio in cervical lymph nodes (P<0.05, respectively).4. Topical application of0.1%dexamethasone eye drops significantly increasedMHC-II+CD11c+cells ratio in peripheral blood (P<0.01), significantly reducedMHC-II+CD11c+cells ratio in spleen (P<0.05), significantly reduced MHC-II+CD11c+ cells and MHC-II+CD86+cells ratio in cervical lymph nodes (P<0.05and P<0.01,respectively).5. Topical application of0.4%rapamycin eye drops significantly reduced increasedMHC-II+CD11c+cells ratio in peripheral blood and cervical lymph nodes (P<0.05,respectively); significantly increased MHC-II+CD86+cells ratio in peripheral blood(P<0.01), significant reduced MHC-II+CD86+cells ratio in cervical lymph nodes(P<0.01).6. Topical application of1%cyclosporine A eye drops significantly reducedMHC-II+CD11c+and MHC-II+CD86+cells ratio in peripheral blood and cervical lymphnodes (P<0.05and P<0.01, respectively); significantly increased MHC-II+CD86+cellsratio in spleen (P<0.05).7. For Elisa examination of peripheral blood serum, compared with the control group,there were no significant differences for IL-2、IL-12、IFN-γ、TGF-β1and CTLA-4levels in5experimental groups (P>0.05, respectively).8. For PCR examination, compare with the control group, IL-2mRNA and IFN-γmRNA expression in1%cyclosporine A eye drops group decreased significantly(P<0.05, respectively); TGF-β1mRNA and CTLA-4mRNA expression in0.5%S1P1eye drops group increased significantly, IL-12mRNA expression reducedsignificantly.9. For mouse corneal graft H&E staining and immunohistochemical examination: incontrol group, there was significant edema and thickening, inflammatory cell wereinfiltrated in the graft epithelium and stroma, lots of CD86+DCs and MHC-II+DCsproliferated and infiltrated; in0.5%S1P1eye drops group, there was epithelial edema,mild thickening of the stroma, the distribution of more inflammatory cells in the graft,there were a few CD86+DCs and MHC-II+DCs proliferation and infiltration in graftepithelium; in0.1%dexamethasone eye drops group, there was corneal epithelialedema, stromal thickening edema, there were more inflammatory cells in the graftstroma, there were little CD86+DCs and MHC-II+DCs proliferation and infiltrationin graft; in0.4%rapamycin eye drops group, there was moderate edema in epithelium,no significant stromal edema, more inflammatory cells were seen within the graft epithelium and stromal, there were some CD86+DCs and MHC-II+DCs proliferationand infiltration; in1%cyclosporine A eye drops group, there was corneal epithelialedema in graft, no obvious stromal edema, there were a few inflammatory cells incorneal endothelium, there was little CD86+DCs and MHC-II+DCs proliferation andinfiltration.10. For0.5%S1P1eye drops combination experiments, the MST of corneal graft incontrol group was16.5±2.3days (n=8); the MST of corneal graft in0.1%dexamethasone eye drops group was32.7±4.0(p<0.01, n=8); the MST of cornealgraft in0.5%S1P1+0.4%rapamycin eye drops was31.8±3.8days (p<0.01, n=8); theMST of corneal graft in0.5%S1P1+1%cyclosporine A eye drops was33.1±3.9days(p<0.01, n=8).11. For0.5%S1P1eye drops drug combination experiments, compared with thecontrol group,0.1%dexamethasone eye drops group,0.5%S1P1suspension eye dropscombined with0.4%rapamycin group,0.5%S1P1suspension eye drops combinedwith1%cyclosporine A eye drops group prolonged mean graft survival timesignificantly (P<0.01); among three treatment groups, the MST in0.5%S1P1suspension eye drops combined with1%cyclosporine A eye drops group was longerthan that in0.1%dexamethasone eye drops group, and the MST in0.1%dexamethasone eye drops group was longer than that in0.5%S1P1suspension eyedrops combined with0.4%rapamycin group, but there was no significant differenceamong three groups (P>0.05).Conclusion:1. In the mouse corneal allograft transplantation model, topical0.5%S1P1eye drops,0.4%rapamycin eye drops and1%cyclosporine A eye drops could significantlyprolong the MST of corneal grafts, but there was no significant difference among thethree experimental groups.2. Compared with above three experimental groups,0.1%dexamethasone eye dropsbetter prolonged mouse MST of corneal grafts significantly. 3.0.5%S1P1eyedrops significantly increased the mature DCs ratio in peripheralblood, reduced mature DCs ratios in cervical lymph nodes;0.5%S1P1eyedrops couldsignificantly reduce the antigen presenting efficiency of DCs.4. Compare with control group, there was no significant difference for IL-2, IL-12,TGF-β1、IFN-γand CTLA-4levels in peripheral blood serum in0.5%S1P1eyedrops group.5.0.5%S1P1eye drops could significantly up-regulate TGF-β1and CTLA-4mRNAexpression and down-regulate IL-12mRNA expression in corneal grafts.6.0.5%S1P1eye drops could significantly reduce DCs in the corneal stromal andendothelium.0.5%S1P1eye drops significantly down-regulated mature DCsexpression in corneal graft.7. In allogeneic mouse corneal transplantation combined regimen,0.5%S1P1suspension eye drops combined with1%cyclosporine A eye drops group prolongedthe MST most in mice, the two drugs had synergistic immunosuppression effect.8.0.5%S1P1suspension eye drops combined1%cyclosporine A eye drops could bealternative choices instead of0.1%dexamethasone eye drops for anti-allogeneiccorneal graft rejection in mice. |
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