| Keratoplasty is a main therapy method. Majority keratopathy patients may recover visual acuity through keratoplasty, this operation have been a principal means nowadays. But the development of this technology mainly relies on reform of technology of cornea preserved. So far, with improvement of equipment and technology, the method of corneal conservation are divided into viable preservation and non-viability preservation according to activity of corneal endothelial cell (CEC ). Besides there is the preservation of short-time, intermediate, long-time according to the time of preservation. In these methods, there are some keeping-time short so that should not satisfy the demand of clinic; some are contaminated easily; and some are wilderness questions needing to solve. Only this profound hypothermia preservation not only shortage life may lengthen out to years but also preservative materials are fineness and cytoactive approximation fresh cornea. After these corneas are used to penetrating keratoplasty, their effect are similar with those fresh corneas. Otherwise, up-to-date lab of tissue engineering is energetic developing, vitrification conservation has been emerged. This technology has been investigated generally a abroad but blank in the domain at home.PurposeThe purpose of this experiment is to desire to find an optimal condition of activity of corneal endothelial cell through long-time cryopreservation. This forte is incomparable in above methods and it is superlative ideal means of conserve cornea at eye bank now. Material and methodsIn the experience we selected 75 clearing rabbits and each rabbit was excised both corneal buttons. Among the rabbits 3 rabbits were selected control group, the rest 147 rabbits were selected experimental group according to the random digits table. There were 3 cornel buttons selected in each experimental group. Corneal button firstly were pre-disposal treatment ,then the corneal buttons were frozen in different phase and different controllable-rated freezer to -80℃; they were stored at the temperature(-196℃) of liquid nitrogen. After gas embolism executes the rabbits, these bulbs of eye of rabbits were extirpated integratedly and were took into the sterile room quickly to pre-emergency. The corneal buttons with a 1～2mm rim of sclera were cut and put into a cornea container and added into 2ml group one liquid and were soaked for 10min at 4℃. Then they were soaked into the other different concentration preservative solutions respectively for 10min and then they were transferred into programmed freezer up to -80℃. Eventually cornea containers with corneal buttons were transferred into liquid nitrogen. After some time, these cornea buttons were took out of the liquid nitrogen and were thawed in the 40℃water bath 2min. Then those preservative solution was removed from the container of cornea and the corneal buttons were eluted Dimethylsulfoxide (DMSO) in 25% Fetus Blood serum (FBS) solution for 10min. Lastly FBS solution was removed. Dyeing and fixation, 0.4% stock solution of Alizarin red S (ph=4.2) was dropwised on the corneal endothelium to dye 3min then saline normal poaching three times; 0.25% solution of Trypane blue was dropwised on the corneal endothelium to dye 2min then saline normal poaching three times; 2.5% solution of glutaraldehyde fixed each corneal buttons for 10min then saline normal poaching three times. In the end morphology observation, investigate the survival rate of each corneal endothelium under the microscope.ResultsControl group were not frozen and corneal endothelium were evidently line up in order, tight junction, under the microscope. Corneal endotheliums present red hexagon adumbration with dual staining of a 0.25% solution of trypane blue and 0.4% stock solution of alizarin red S(ph=4.2) and are not dyed blue cell nucleus. Corneal endotheliums are integrated in morphology and uniformity in size at the whole eyesight. But the experiment group that through different freezing velocity discovered that intercellular space are widen under the microscope. Although some cells are still arranged regularity, cellular adumbration is irregular shape. The rest various kinds methods display untidiness, appearance not regular, cellular adumbration not exist. There are many blue cell nucleus and cell fusion still may be found, moreover some cell are loss and expose parts of the membrane of Descemet even cell fusion flaky merely remain naked nucleus.Through contrast research we found that the rate was -2.0℃/min in the first phase and the rate was -5.0℃/min in the Second phase, the corneal endothelium survival rate were best. There were significant difference (p<0.05).ConclusionsMultiplicity of cornea cryopreservation and expensive of the programmed freezer have confined the development of more eye banks. Considerable experiments have proved that endothelial cell survival rate of cornea could achieve above 70% in the base of rigorous program and rigorous procedure and this is significant to eye bank.
|
PDF Full Text Request