| OBJECTIVEKeratopathy is a kind of common and frequently encountered disease, and the main reason for blindness. Corneal transplantation is the important operation to recover the eyesight, and also main treatment for corneal disease. Because cornea is immune privilege tissue, corneal transplantation is the most successful form of the organ allograft. However immune - mediated rejection is still the leading cause of corneal allograft failure. Current immunosuppressive drugs used to prevent or treat corneal graft rejection include corticosteroids, cyclosporin A and FK506, which greatly improve achievement rate of corneal transplantation and long - term survival rate of the corneal allografts, but rejection in some patients still can not be suppressed. Nowadays, searching high efficacy, low toxic-ity immunosuppressant is always a chief problem to prevent or treat corneal graft rejection. Rapamycin is a macrolide antifungal antibiotic produced by Strepto-myces hygroscopicus and was later found to have immunosuppressive qualities. It has been shown to exert potent immunosuppressive effects in a variety of in vitro/in vivo transplantation as well as autoimmne disease models, and it is a new immunosuppressive agent that has good therapeutic effect, low toxicity, without nephro - and neurotoxic properties. But there are only superficial papers about rapamycin in low - risk corneal transplantation models, and it is indicated that RPM could markedly prolong the survival time of corneal allografts by systemic use. In this research, in order to avoid toxicity of systemic administered rapamycin, we intend to evaluate the safe concentration of rapamycin ophthalmic solution by local application on ocular surface, study the immunosup-pressive effect on allogeneic comeal transplantation in rat models, observe the synergistic effect combined with cyclosporin A, and investigate the mechanism in suppressing immunologic rejection in rat corneal transplantation. We also intend to provid new combined therapy in preventing or treating immunologic rejection of corneal transplantation in clinic.METHODS1. To study the stimulation in ocular region and the toxicity of rapamycin ophthalmic solution. 20 SD rats were randomly divided into 4 groups, and 5 rats in each group. A control(ophthalmic solution solvent), B (0. 1% rapamycin ophthalmic solution), C (0.2% rapamycin ophthalmic solution), D (0.4% rapamycin ophthalmic solution). Clinical observation was taken to evaluate the eye stimulation of each ophthalmic solution by the standard of reaction to stimulated eye. General body state and eye damage of the animals were observed, and the pathologic changes of the corneas, hearts, livers, spleens, lungs and kidneys were measured by light microscope.2. To evaluate the pharmacodynamics of rapamycin ophthalmic solution in immunological rejection after allogeneic corneal transplantation in rats by local application. SD rats were used as recipients, and Wistar rats were used as donors. Animals were randomly divided into 6 groups. A control (ophthalmic solution solvent lOOui) , B (0.05% rapamycin ophthalmic solution 100|xl) , C (0. 1% rapamycin ophthalmic solution 100(xl) , D (0.2% rapamycin ophthalmic solution 100ui) , E (1% cyclosporin A ophthalmic solution 100|xl), F (0.2% rapamycin ophthalmic solution 50 ul and 1% cyclosporin A ophthalmic solution 50|xl). The animals were given the drugs 4 times every day. Treatment was begun on the second day after operation, the animals were administered until rejection, and the experiment was ended on the 42d after operation. The corneal grafts were inspected by slit - lamp microscope and the corneal survival time was recorded.3. To study the mechanism of rapamycin ophthalmic solution in immunological rejection after allogeneic corneal transplantation in rats by local application.SD rats were used as recipients, and Wistar rats were used as donors. Animals were randomly divided into 4 groups. A control ( ophthalmic solution solvent IOOjulI) , B (0.2% rapamycin ophthalmic solution lOOui) , C (1% cyclosporin A ophthalmic solution lOOfjd) , D (0. 2% rapamycin ophthalmic solution 50jxl and 1% cyclosporin A ophthalmic solution 50jxl). The animals were given the drugs 4 times every day. Treatment was begun on the second day after operation. The pathologic changes of the corneas were measured by light microscope, and IL - 13, IFN - -y and PFP mRNA of the corneas were examined by quantitative reverse transcription polymerase chain reaction ( RT - PCR) on the 14d after operation.4. To study the immunosuppressive effect of rapamycin ophthalmic solution on immunological rejection after high - risk corneal transplantation in rats. SD rats with corneal vascularization were used as recipients, and Wistar rats were used as donors. Animals were randomly divided into the following groups, A control (ophthalmic solution solvent lOOfxl), B (0.2% rapamycin ophthalmic solution lOOfil) , C (1% cyclosporin A ophthalmic solution lOOui), D (0.2% rapamycin ophthalmic solution 50juJ and 1% cyclosporin A ophthalmic solution 50jxl). The animals were given the drugs 4 times every day. Treatment was begun on the second day after operation, and the animals were administered until rejection. The grafts were inspected by slit - lamp microscope and the corneal survival time was recorded, and the pathologic changes of the corneal grafts were measured by light microscope.RESULTS1. The marks of the eye stimulated reaction by locaU}? using the ophthalmic solution solvent, 0. 1% rapamycin ophthalmic solution and 0. 2% rapamycin ophthalmic solution were 0, and 0.4% rapamycin ophthalmic solution was 1. 20. There was no fluorescein sodium stain on the corneal epithelium. Animals in each group had normal food and drink, and the weight of them was all increased. There was no damage in conjunctiva, cornea, iris, crystalline lens and retina. The histopathological findings showed that there were no abnormal chan-ges in the corneas, hearts, livers, spleens, lungs and kidneys.2. The survival rate of the corneal grafts in each treated group was statistically higher than that in the control group (P <0.01). Group D was superior to group B and group C (P<0.01;P<0.05). Group F was superior to the other treated groups( compared with group D, P <0.05;compared with the other treated groups,P < 0.01). Group B, group C and group D had no statistical difference compared with group E ( P > 0.05).3. The histopathological findings showed that the inflammatory cells, neo-vascularity and edema of the corneal grafts in each treated group were significantly fewer than that in the control group on the 14d after operation, and there were no inflammatory cells and neovascularity in the centre of the grafts. IL -1 p, IFN - -y and PFP mRNA were weakly expressed in normal corneas, but there was a significant overexpression in the control group which the allografts rejected within 14d after operation (P < 0. 01). In each treated group, IL — 1 (B, IFN - 7 and PFP mRNA were weakly expressed compared with the control group ( P < 0.01).4. The mean survival time of the comeal grafts in each treated group of high - risk corneal transplantation was 16. 67 ± 1. 63d, 15. 50 ± 2.43d and 21. 33 ±2. 94d respectively, which statistically prolonged compared with the control group (P<0.01). The combination therapy (group D) was significantly superior compared with group BorC (P<0.01). There was no statistical difference between group B and C (P >0. 05). The result of survival rate comparison in the groups was coincidental with that of survival time comparison. The histopathological findings showed that the inflammatory cells, neovascularity and e-dema of the corneal grafts in each treated group were significantly fewer than that in the control group on the 14d after operation, and there were no inflammatory cells and neovascularity in the centre of the grafts in combined treated group.CONCLUSIONS1. There are no stimulation and toxicity by local application of ophthalmic solution solvent, 0. 1% rapamycin ophthalmic solution, 0.2% rapamycin oph-thalmic solution, and 0.4% rapamycin ophthalmic solution.2. It is indicated that 0.05% , 0.1% and 0. 2% rapamycin ophthalmic solution could markedly prolong the survival time of corneal allografts by inhibiting graft rejection, the suppressive effect increased along with high concentration, and the suppressive effect of 0. 2% rapamycin ophthalmic solution was the big-gist. The combined therapy of 0. 2% rapamycin and 1% cyclosporin A ophthalmic solution produced synergistic effect, which was superior to rapamycin or cyclosporin A alone.3. It is indicated that 0. 2% rapamycin ophthalmic solution could markly suppress inflammatory cells infiltration and the expression of IL - 1 (3, IFN - *y and PFP mRNA, which must be one of the mechanism of it' s suppressive effect in corneal transplantation.4. It is indicated that 0. 2% rapamycin ophthalmic solution could markly prolong the survival time of corneal grafts in high — risk corneal transplantation. The combined therapy produced synergistic effect, and was superior to rapamycin or cyclosporin A alone.
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