Experiment Study On The Correlation Of Penetrating Keratoplasty Rejection And Cornea Preservation Methods

Experiment study on the correlation of penetrating keratoplasty rejection and cornea preservation methodsOBJECTIVEKeratopathy is a kind of common and frequently encountered disease and the main reason for blindness nowadays. Corneal transplantation is the most effective method to cure keratopathy. Eye bank and corneal preservation technology are the basis of the corneal transplantation. However, the source of the corneal grafts are deficit and maldistribution, It is not satisfied the demand on the multitudes of corneal transplant patients and dealing with emergency operation. So, Establishing effective eye bank, deeply investigating corneal preservation, prolonging corneal life, utilization limited grafts, providing high-quality grafts anytime is a very important topic. In view of the limit of short-term preservation, we can contribute to corneal transplantation if we make intermediate and long-term preservation. Corneal reject reaction is the main cause on the defeat of corneal transplantation. It is a multiple factor, extremely complicated and highly regulative process. How to lower the corneal reject reaction has become many scholars main research topic. In this research, in order to offer high-quality grafts for clinical use and establish foundation to make use of them, we intend to search for simple and safe intermediate and long-term preservation methods to estimate corneal endothelium viability after different preservations. To understand the relation between the penetrating keratoplasty rejection and the time of preserved cornea, we aim at the observation on the corneas used transplantation of different preservation methods. To explore a new way to increase achievement ratio and decrease rejection on corneal transplantion, We also assay qualitative and quantitative content of cell factor and adlhesion molecule and analyze the reason through the methods of pathology and immunology.METHODS1. Simple and easy intermediate and cryopreservation methods in microcomputer monitoring procedure were established. Thirty Wistar rat eyeballs were randomly classified into 3 groups in the moist chamber storage, intermediate and long-term preservation. The differences of the viability of corneal endothelial cells after different preservation methods were compared dyeing with trypan blue and alizarin red.2. The 30 Wistar rats and 15 SD rats were used to establish the animal models of penetrating keratoplasty rejection and were randomly divided into 3 groups. SD rats were used as recipients and Wistar rats were used as donors. The different time preservated cornea donor of Wistar rats were used separately in three groups. The penetrating keratoplasty rejection index (RI) in postoperation, means survival time (MST) of corneal grafts and histopathological changes were analyzed. Tissues of corneal transplants from the three groups of rats were cut and treated with immunohistochemistry and HE staining. The relationship between the intensity of immunity rejection and the severity of inflammation was monitored. The levels of TNF-a in serum and aqueous humor were determined dynamically by enzyme linked immunosobent assay (ELISA) after 10 days.RESULTS1. The endothelial cell density and activity ratio were similar after different preservation methods. The vital staining showed that there were no significant among three preservation methods on the endothelial cell density (cell/mm~2) and ESR%. (the moist chamber storage, cell/mm~2 2729.5±158.0, ESR%88.9±3.5; intermediate preservation cell/mm~2 2646.8±217.2, ESR% 86.94±2.7; cryopreservation cell/mm~2 2706.2±184.2, ESR% 87.3±3.3). So, as long as if only the committed steps were reasonable, the endothelial cell density was not obviously decreased and activity ratio was above 85%.2. The MST was 10.4±1.70days in short-term-preserved group, 12.9±1.81days in medium-term-preserved group and 16.1±2.57days in long-term-preserved group. The MST in the long-term-preserved group was evidently prolonged, showing a significant correlation among three groups (P＜0.01). The sections with HE staining revealed that the severity of immunological rejection and histoleucocyte and homeocyte infiltration and new vessels inⅢgroup was reduced compared with that ofⅠ,Ⅱgroup after 10 days of keratoplasty.Ⅱgroup was reduced compared with that ofⅠgroup. Immunohistochemistry staining revealed that the intensity of ICAM-1 expression inⅢgroup was lower thanⅠ,Ⅱgroup. The levels of TNF-a in serum and aqueous humor inⅢgroup (213.76±19.06pg/mL, 203.17±16.62pg/mL)was evidently lower thanⅠ,Ⅱgroup (269.08±14.28 pg/mL, 240.87±9.28 pg/mL; 336.51±38.50pg/mL, 284.83±28.76pg/mL), showing a significant correlation among three groups (P＜0.01).CONCLUSIONS1. Intermediate and long-term cryopreserved methods are very useful on penetrating ketoplasty.2. The rate of rejection of penetrating keratoplasty in postoperation is decreased and rejection time is delayed in the variant long-term cryopreserved group compared with that of the intermediate and moist chamber group. The variant intermediate group is reduced compared with that of the moist chamber group. ICAM-1 and TNF-a, as significant agent in corneal graft rejection, have positive correlation with rejection.