An Experimental Study On The Effect Of FTY720 On The Immunological Rejection After Corneal Transplantation In Rats

There are approximately 4,000,000 blind patients caused by corneal diseases in China. Corneal transplantation is the most effective operation to regain visual acuity. Although corneal transplantation is the most successful form of the organ allografts, its rejection rate range from 10% to 30%. Factors contributing to high-risk keratoplasty including vascularized cornea, prolonged inflammation, large size keratoplasty, and rekeratoplasty, and the rejection rate can reach at 60% above, which is similar to other organ's transplantations. Generally speaking, immunological rejection is the crucial factor that contributes to the failure cornea transportation. How to lower and prevent the rejection rate after this operation is the key problem, however, to search for a high-effective, lower-toxic immunosuppressive agent is the vital step to solve the problem above.Nowdays, a new high-effective immune depressive therapy with CsA has been generally utilized in clinic. But the demerits such as toxicant or adverse reaction for liver and kidney, high blood pressure, diabetes, toxic nervous system, secondary infection and tumor caused by immunosuppressant haven't been fundamentally overcomed. During the past two decades, to develop a neotype and low-toxic immunosuppressive agent is the issue that many related scholars show great concern.FTY720 is the antibiotics elements ISP-1 which is extracted from Chinese Caterpillar Fungus by chemical modification; It's really a synthetic drug that takes immunosuppressive action notably. The clinical study shows that FTY720 does have perfect immunosuppressive activity in rodents animals' liver, kidney, skin, heart, pancreatic islet and homotransplantations and dog's liver, kidney. FTY720 can obviously prolong the transplanted receiver and transplanted giver' life and takes on dose dependant. We seldom hear the report that FTY720 is applied to cornea transplantation. Unlike traditional immunosuppressant the mechanism of action of FTY720 is unique. Its Pharmacologic action functions quickly and can reduce peripheral blood lymphocyte and T cell population increasingly, which return lymphocyte into periphery lymph node, mesenteric nodes, and payer's nodes. The process can be reversible. This will isolate lymphocyte from its sublevel lymphatic tissue, and protect transplant away from the lymphocyte's infiltration and attack. However, it's rare that we read the report on the lasting effect of blood-lymph after corneal transplantation in rat by using FTY720.FTY720 and routine immunosuppressant, they both have joint action. A little CsA and FTY720 can prolong transplant's life for rodent's animals and primate animals, therefore, it can decrease the side effect caused by CsA.Unlike CsA immunosuppressant, the organism might not infect when using FTY 20. Proper amount of FTY720 will not give rise to toxic liver, kidney, neurotoxicity, abnormal lipid metabolism, over-immune.The experiment aims at observing corneal transplantation of graft rejective reaction after applying FTY720 to rat's whole body, monitoring the amount of outer peripheral blood lymphocyte dynamically and expression of corneal implant vascular endothelial growth factor and CD25(IL-2R) via establishing penetrating corneal transplantation model, furthermore it will probe the function and organism of FTY, providing evidence for corneal transplantation of graft rejective reaction of clinical therapy.Objective:1. Observe from the aspect of pharmacodynamics: establish homogeneity variant penetrating corneal transplantation model of rats. Observing the effect of immunological rejection of implant after intraperitoneal injection with different amount of FTY720 dose ,and compared its effect with the 1% cyclosporine A(CsA), appraising it effect, providing dosage regimen for clinic.2. the probe of organism: a)corneal transplantation rat after the intraperitoneal injection with different amounts of FTY720, detecting rat's changes of peripheral blood lymphocyte at different time b)during the period of implant's acute cellular rejection, inspect different expressions between vascular endothelial growth factor of blank control group and the group using different amount of FTY720 dose and interleukin2 recipient (IL-2R). Those results can help us study the possible mechanism of action through the classical immune route of corneal transplantation graft rejective reaction.Methods:1. To take the SPF average Wistar rat as donators, SD rat as recipient, set up the penetrating corneal transplantation model of same variant, randomly divide them into group A, group B, group C and group D. Group A: negative Control; group B: 1%CsA; group C: FTY720 intraperitoneal injection (0.2mg/kg/d);group D: FTY720 intraperitoneal injection (0.6mg/kg/d).Each group was applied to dose from the first day to fourteenth day. Twelve days after operation, observe each group's transparency, the degree of edema, the degree of new vessels, the index of transplantation rejection, and corneal rejection by slit lamp microscope. Adopt SPSS12.0 for window statistics software as a way of statistical analysis, express data with mean±standard deviation, keep record of implant's survival time, draw Kaplan-Meier curve, compare with Long-rank test; Apply the One-way ANOVA on all groups' clinical parameter twelve days later after operation. Besides the method of LSD will be applied to all groups, If result should be P≤0.05, it shows the statistical significance.2. To take the SPF average Wistar rat as donators, SD rat as recipient, set up the penetrating corneal transplantation model of same variant, randomly divide them into group A, group B. group A: FTY720 intraperitoneal injection (0.2mg/kg/d); group B: FTY720 intraperitoneal injection (0.6mg/kg/d). Each group was applied to dose from the first day to fourteenth day. Collect rat's peripheral blood by cutting part of rats' tails after operation, before operation, at the period of dose using and after dose cease. Employ SPSS12.0 for window statistics software for statistic analysis, express data with mean±standard deviation, keep record of quantity of peripheral blood lymphocyte from all groups of rats at different time. Compare with the analysis of variance of duplication measured data. If result should be P≤0.05, it shows the statistical significance.3. To take the SPF average Wistar rat as donators, SD rat as recipient, set up the penetrating corneal transplantation model of same variant, randomly divide them into group A, group B, and group C. group A: negative Control; group B: FTY720 intraperitoneal injection (0.2mg/kg/d);group C: FTY720 intraperitoneal injection (0.6mg/kg/d). Twelve days after operation, take the implant of corneal, manufacture paraffin section and HE dyeing, observe pathology change of all groups' corneal implant by light microscope, and inspect distribution and expression of VEGF and IL-2Rin all groups' corneal tissue by immunohistochemistry dyeing (SABC method).Adopt SPSS12.0 for window statistics software as a way of statistical analysis, express data with mean±standard deviation. Utilize the One-way ANOVA on all groups. If result should be P≤0.05, it shows the statistical significance.Result:1. Average survival time of each implant: blank contrast group (11.123±1.959)d, 1%CSA eye drop application group(16.125±2.532)d, 0.2mg/kg/d peritoneal injection FTY720 group(35.000±26.490)d, 0.6mg/kg/d intraperitoneal injection FTY720 group(46.750±33.148)d. The difference of all groups' survival time has statistic significance(X2=53.746, p=0.000), the difference of all groups has statistic significance(averagely p=0.000).2. period of acute cellular rejection, the quantity of implants' new vessels of 0.2mg/kg and 0.6mg/kgFTY720group are less than blank contrast group (respectively be p=0.001, p=0.000);In general, from the aspect of anti-rejection, the effect of group RI is better than that of group cyclosporine A drop application (respectively P=0.002, P=0.000).3. FTY720's effect on peripheral blood lymphocyte of corneal transplantation rat.(1) After intraperitoneal injection FTY720, peripheral blood lymphocyte decreased increasingly. Five hours after applying to drug peripheral blood lymphocyte decreased by lowest amount. Group 0.2mg/kg lower its amount to 35.2% compared to operation before. Group0.6mg/kg group lower its amount to 17.4% compared to operation before.(2) During the period of dose application, each group's PBL maintains considerably low level. During the whole observation period, PBL of Group B is less than group A (F=43.536, p=0.000).(3) After ceasing drug the quantity rise. Two weeks after ceasing drug the amount of PBL of group 0.2mg/kg rise to its former level(0.444).Four weeks after ceasing drug the amount of PBL of group 0.6mg/kg rise to its former level(p=0.089).4. The pathology of corneal implant during acute rejection period and immunohistochemisty.(1) HE dyeing indicates: implants of blank contrast group thicken prominently, large quantities of histoleucocyte and lymphocyte infiltrate, and neovascularization forms. The thickness of implant of rats that utilizing FTY720 in the whole of their body is normal, no obvious inflammatory cell infiltrate, no neovascularization.(2) Immunohistochemistry shows: masculine cell population IL-2R and different amount of implant VEGF of group FTY720 are less than blank contrast group. (p＜0.05).Conclusion:1. Applying FTY720 to the rats' whole body can prevent corneal graft rejection effectively, and prolong the survival time of implant. With the increasing dose the effect would be better.2. The side effect of FTY720 remains at lowest level, the range of therapy is wide; therefore it's safe to use it with comparatively large amount of dose.3. FFY720 peritoneal injection with 0.2mg/kg or 0.6mg/kg can both inhibit the forming of new vessels.4. FTY720 can reduce the expression of VEGF an IL-2R protein implant at acute period, furthermore it will diminish the quantity of peripheral blood lymphocyte, hold back the possibility that the inflammation cell infiltrate the transplant. Maybe, this is the organism that FTY720 prevents rejection.