Research And Application Of Reformed Cornea Preservation Liquid In Mid-Term

Objective: 1. To research on a kind of mid-term preservation liquid that can be applied into cornea storage in eyebank.2. To discuss the function and effective concentration of bFGF and SH in mid-term cornea preservation liquid.3. To assess the effect and application value of the preservation liquid by preliminarily observing the experiment on live animals and clinic operation cases.The experiment can be divided into three parts:(1) Research on mid-term cornea preservation liquid and the protective function of Corneal Endothelial Cells;(2) Application of reformed mid-term cornea preservation liquid to cornea storage by experimenting on live animals;(3) Preliminary clinic research on the application of reformed mid-term cornea preservation liquid to cornea storage.Methods: (1) Research on reformed mid-term cornea preservation liquid: To make up cornea preservation liquid by basal medium of MEM and M-199 as its main constituent and six percent of dextran, HEPES buffer system and antibiotic as its adding part. To add SH and bFGF into it and divide it into four experimental groups, say, A (SH0.03% bFGF 20ng/ml), B(SH 0.05%bFGF 50ng/ml), C(SH0.1% bFGF 100ng/ml) and D(SH 0.2% bFGF 200ng/ml). Besides, group E should be included (not adding SH and bfGf into it). Preserve rabbits'corneas at 4℃respectively at 3, 5, 7, 10 and 14 days. After risen to (30-40)℃, detect vitality of corneal endothelial cells with slit-lamp examination, trypan blue-alizarin red staining, pathological section of corneal tissue and scanning electron microscope so as to assess the preservation quality. During the period of the storage of preservation liquid, detect bacteriology regularly, and assess the safety of biology.(2) Experiment on live animals by applying reformed mid-term preservation liquid to corneal storage: Use preservation liquid of group D (SH 0.2% bFGF 200ng/ml) to preserve rabbits'corneas for 3, 5, 7 and 9 days respectively (two pieces for each period), then apply the preservation liquid to variant rabbit penetrating keratoplasty (one rabbit for each). Observe the transparency of preserved implant, reaction of anterior chamber and suture line, and then give assessment of the effect of the operation.(3) Preliminary clinic research on the application of reformed mid-term cornea preservation liquid to cornea storage: Use preservation liquid of group D (SH 0.2% bFGF 200ng/ml) to preserve donor's corneas. After one to four days'preservation, apply the preservation liquid to clinic penetrating keratoplasty. Observe the transparency of preserved implant, acuity of vision and complication after seven to fourteen days, and then give assessment of the clinical effect.Results: (1) The first part: Through observing the general morphology and slit lamp, we find that the preservation liquid of each group (10 pieces for each) are clear, transparent with no stuffy and no turbidness. In a short period, the corneas in each group are transparent. There is no hydropsia and thickening. Structures of each layer are clear. As time passes by, hydropsia has appeared in group A, B and C, and the degree of transparency has reduced. But corneas of group D are transparent, and no hydropsia has appeared. The degree of hydropsia in Group E is the most serious and structures of all the layers are almost dim. In the advanced stage, group A, B and C are in the state of hydropsia and become dim. Corneas of group D are transparent with light hydropsia and the substantia propria layer becomes slightly thickening. Group E has appeared serious hydropsia with fuzzy structures. Group D owns the highest percentage of trypan blue-alizarin red staining competent cells. Group E (control group) is the worst. We can see that changes among the five groups show the obvious variability by observing the pathological section of corneal tissue. Every experimental groups are better than the control group, among which group D is the best. Observing the seven-day-preserved corneas in group D through ultrastructure, we can see microvilli and the basically compete cell junction. Besides, there is no obvious breakage of the cell membrane. (2) The second part: Performing the corneal transplantation of live rabbits by using the rabbits'corneas which have been preserved for 5, 6, 7 and 9 days respectively (two rabbits for each). Observing the corneal implants for thirty days after the operation, we can find that all the corneal implants operation has been confirmed. (3) The third part: Preserving the donor's cornea for one to four days, and then performing the penetrativity corneal transplantation. By observing thirty-two cases, we can find that the corneal implants are transparent, and the patients'eye sight has improved in various degrees. During the hospital stay, we find no complication such as immunological rejection, corneal new vessels, secondarg glaucoma or endophthalmitis, which has proved that the operation is safe and effective. Conclusions: Making up mid-term cornea preservation liquid by the following constituents: MEM and M-199 as its major component, 0.2% SH, 200ng/mlbFGF, six percent of dextran, HEPES buffer and antibiotic, which can obviously improve the quality of corneal preservation and extend the time for preservation. The experiment on live animals and the preliminary clinic observation have proved that the preservation liquid can effectively preserve the corneal implants for seven to ten days with good result. The home-made preservation liquid with low cost is safe and asepsis, and easy to make. It can be applied into the cornea preservation in bank.