The Role Of Basic Fibroblast Growth Factor And Carnosine On The Rabbit Corneal Endothelial Cells Preserved In Mid-term Preserveing Medium

Objective: To investigate the role of basic fibroblast growth factor (bFGF) and carnosine on corneas preserved in mid-term preserving medium, meanwhile, to explore the availability of carnosine applied in mid-term preserving medium,in order to improve the medium of corneal mid-term preservation.Methods:(1):To make up cornea preservation liquid by basal medium of MEM as its main constituent and 1% dextran-40,2.5%CS,HEPES buffer system and streptomycin as its adding part.(2)To add bFGF and carnosine into it and divide it into three experimental groups, say, A(bFGF 20 ng/ml), B(carnosine 5g/L) and C(bFGF 20 ng/ml, carnosine 5g/L).Besides,group D should be included(not adding bFGF and carnosine into it).Preserve rabbits'corneas at 4℃respectively at 3,7and 14 days.After risen to(30-35)℃,detect vitality of corneal endothelial cells with trypan blue-alizarin red staining, pathological section of corneal tissue, scanning electron microscope(SEM) and transmission electron microscope(TEM)so as to assess the preservation quality.(3)During the period of the storage of preservation liquid, detect bacteriology regularly, and assess the safety of biology.Results:1. General morphology of corneas: In the early stage,the corneas in each group are transparent.There is no hydropsia and thickening. As time passes by, hydropsia has appeared gradually in all groups, and the degree of transparency has reduced. The degree of hydropsia in Group D is the most serious.In the advanced stage, Corneas of group A and C are transparent with light hydropsia.Group D has appeared serious hydropsia and thickening.2. Trypan blue-alizarin red staining: At the 7th and 14th days of preservation, group A and C owns the highest percentage of trypan blue-alizarin red staining competent cells.Group D is the worst.3. Pathological section of corneal tissue: At the 7th and 14th days of preservation, compared with the control group, the corneal Structures of experimental groups are clear, the degree of hydropsia in stroma are slight. The corneal epithelial cells attached closely to the Descement's membrane.4. Ultrastructural morphology: SEM results:⑴At the 14th days of preservation, in experimental groups we could see that the shape of CECs and the connections between CECs were normal, and there were some microvilli on the surface of CECs;⑵But in control group, the necrosis of CECs was pervasive, the residual CECs were obviously swelling and the connections of CECs had obvious breakage. TEM results:⑴At the 14th days of preservation, in experimental groups, the cell body of CECs was increased slightly. There are few vacuoles in cytoplasm, the mitochondria were slightly swollen and the endoplasmic reticulum were slightly dilated.⑵But in control group, the degeneration and necrosis of CECs was obvious, There are a lot of vacuoles in cytoplasm, the mitochondria were severely swollen and the endoplasmic reticulum were severely dilated, karyopycnosis were emerged..5. During the preserved period, the preserved solution of all groups were transparent, there was no turbidness. pH value was maintained at 7.2-7.4, the bacterial culture was negative. Conclusion:Addition of bFGF(20 ng/ml) and carnosine(5g/l) in corneal storage medium plays a significant role in maintaining the viability of the preserved CECs. We can apply bFGF and carnosine to corneal mid-term preservation. Our experiment also suggested it will be more effectively to addit bFGF in mid-term preserveing medium than carnosine.