Experimental Study On The Effect Of Cryopreservation To Corneal Antigenicity

Objective: Corneal transplantation is the most successful organ transplantation at present. Corneal epithelia and hypothallus only express a small quantity of dendritic cells. Blood vessel and lymph node are absent in the cornea, which are main factors of corneal immune privilege. However, immune rejection is the chief cause for the failure of corneal transplantation. Cryopreservation plays an important role on that it can provide cornea for clinical use when the source of cornea is limited. It has been proved by the previous experimental study and clinical use that the viability of corneal endothelia after cryopreservation can be maintained and fit for keratoplasty. It is not concluded whether the antigenicity of the cryopreserved cornea is changed or not. The purpose of this research is to study the change of corneal antigenicity after cryopreservation in liquid nitrogen and its mechanism.Methods: The corneas were trephined from screened fresh pig eyeballs. The diameter of each graft was 13mm. The grafts were put into the cool cryoprotective medium and equilibriumed with simplified four-step method. Some of the grafts were directly cryopreserved in liquid nitrogen , others were gradiently frozen. All of the grafts were preserved for two months. 40 BALB/c mice were randomly divided into 4 groups: the fresh cornea group, the viably cryopreserved cornea group, the directly cryopreserved cornea group and the control group. Except for the control group, the grafts were transplanted into the hypodermis of the mice' thoracic wall. CD4+, CD8+, CD25+, CD71+ T lymphocytes were counted by flow cytometer on 12d after transplantation. The corneal grafts were dissected, imbedded in paraffin and HE stained. The pathological changes were observed with microscope.Results: On 12d after transplantation, grafts of the directly cryopreserved group were translucent, while grafts of the viably cryopreserved and fresh groups were completely clouding. The number of Lymphocyte in the former group was less than that in the latter two groups. Compared with the control group, the numbers of CD4+CD3+/CD3+, CD25+CD3+/CD3+, CD71+CD3+/CD3+ in peripheral blood and the value of CD4+/CD8+ increased in BALB/c mice, while the numbers of CD8+CD3+/CD3+ decreased. There were significant differences between the fresh cornea, viably cryopreserved cornea and directly cryopreserved cornea groups.Conclusions: Different freezing rates lead to different changes of the corneal antigenicity. The cryopreservation which reducing the temperature fast lead to the low antigenicity.