Studies On Rapamycin Eye Drops In Prevention Of High-risk Corneal Allograft Rejection In Rats

BACKGROUNDCorneal transplantation is an important operation of vision reconstruction andone of the most successful organ transplantations.The previous studies show that therejection rate in the low-risk cases is only 10%, while in the high-risk cases is higherthan 70%.Immune rejection is the main reason of allograft failure of high-riskkeratoplasty .There are many high-risk factors such as corneal neovascularization,large size keratoplasty, acceptance of other organ transplantations and so on. Afterhigh-risk keratoplasty patients have poor visual acuity and doctors have no moremethods to deal with them.How to suppress immune rejections is an urgent task forall scientists and doctors.At present immunosuppressants are widely used in clinicalaffairs, but there are few kinds of them.New types of immunosuppressants areneeded.Since the high-risk keratoplasty has such a high rejection rate, it is urgent tofind a new way solve this problem.Rapamycin (sirolimus) was found in 1975 as an anti-fungal medicine.In 1978, itwas reported that rapamycin was a useful immunosuppressive drug.Since then, itbecame hot in immunosuppressive researches. Previous studies showed thatrapamycin could prolong the survival time of transplanted organs such as kindeys, hepars, skin, hearts and so on. Rapamycin was also reported to inhibit rejections aftercorneal transplantations by systemic administration, however rapamycin eye dropswas left untouched. In the studies rapamycin eye drops will be prepared and furtherresearches are to be carried out.PartⅠToxicological study of rapamycin eye drops on rat corneasOBJECTIVETo observe the toxicity of rapamycin eye drops in rat corneas and determine thesafe concentration for experimental and clinical use.METHODSRapamycin was dissolved in castor oil ,and thus 0.5%,1% and 2% rapamycineye drops were prepared.Fifteen SD rats were randomly devided into five groups withnormal saline, castor oil, 0.5% rapamycin eye drops,1% rapamycin eye drops and 2%rapamycin eye drops.Eye drops were applied six times daily and one drop pertime.Rat corneas were checked by slit lamp microscope everyday. Seven days later,histopathological methods were carried out on all right corneas and left corneas wereevaluated under transmission electron microscope.RESULTSNo significant changes were observed among normal saline group,castor oilgroup, 0.5% rapamycin group and 1% rapamycin group. On the fourth day swelledcorneal epithelia was observed in the 2% rapamycin group, and no further damageswere found as time went by. Unregular basal corneal epithelia, wide intercellar space,cellular swelling, engored bioblast, broad endocytoplasmic reticulums were alsofound.CONCLUSION 0.5% and 1% rapamycin eye drops do no harm to rat corneas. Castor oil is agood kind of ophthalmic solution solvent.PartⅡInhibition of rat corneal neovascularization by rapamycin eye dropsOBJECTIVETo study how rapamycin eye drops inhibited cornea neovascularization and toinvestigate the expressions of VEGF and IL-2Rαin rat corneas.METHODSCorneal neovascularization was induced by suture method, and then 12 SD ratswere randomly assigned to two groups: normal saline group and 1% rapamycin group.Drugs were delivered four times daily after operations. Rat corneas were checked byslit lamp microscope everyday.The length of corneal neovascularization wasmeasured at the third, the seventh and the fourteenth day, so the area of cornealneovascularization was recorded. 14 days later the expressions of VEGF and IL-2Rαwere detected with immunohistochemical techniques.All the data were recorded by mean±standard deviation. Repeated MeasureANOVA was carried out (SPSS version10.0). Statistical tests were consideredsignificant when P values were less than 0.05.RESULTSCorneal neovascularization area of normal saline group is significantly higherthan that of 1% RAPA group(F=292.507,P=0.000). Corneal neovascularization areaof 1% rapamycin group was less than that of normal saline group during the period ofthe experiment (t=11.614,8.403,12.638;P=0.000). The expression of VEGF wasfound in corneal epithelia, stroma, endothelia, corneal neovascularization andinflammatory cell in normal saline group. At the same time IL-2Rαexpression was also found. Less expressions of VEGF and IL-2Rαwere found in 1% rapamycingroup.CONCLUSION1% rapamycin eye drops inhibit corneal neovascularization and reduce theexpressions of VEGF and IL-2Rα.PartⅢRapamycin eye drops in prevention of high-risk corneal allograft rejectionOBJECTIVETo study the immunosuppressive effects of 1% rapamycin and 1% Cyclosporin Aeye drops.METHODHigh-risk allograft beds were made in SD rats according to PartⅡ.Penetratingkeratoplasty was performed with Wistar rats as donors. 32 recipient rats wererandomly devided into four groups: normal saline group, 1% Cyclosporin A group,1% rapamycin group and Cyclosporin A & rapamycin group.Treatments began on thefirst postoperative day. Eye drops were applied four times daily and one drop per time.The corneal allografts were examined by slit lamp microscope everyday and rejectionindexes were recorded at the same time.Data were recorded by (?)+s. One-way ANOVA was carried out (SPSS version10.0). Least-significant difference (LSD) was chosen to compare mean CNV areabetween every two groups when homogeneity of variance was accepted andotherwise Tambane's T2 was suitable. Statistical tests were considered significantwhen P values were less than 0.05.RESULTSSignificant differences of survival time among the four groups were found (F=87.372,P=0.000). Graft survival time was 7.9d, 11.3d, 13.6d and 16.5drespectively and statistical difference between every two groups was found (P=0.000).CONCLUSION1% rapamycin eye drops prolong the survival time of corneal allografts inhigh-risk penetrating keratoplasty.1% rapamycin eye drops are more effective than1% Cyclosporin A eye drops.The most effective way to prolong the graft survivaltime is to combine 1% rapamycin and 1% Cyclosporin A eye drops.