Observation On Effect Of Application Of Chondroitin Sulfate On Corneas Preser-vation After Penetrating Keratoplasty

ObjectiveTo investigate the protective effect of chondroitin sulfate（CS）on cornealendothelial cells（CECs）preserved by cryopreservation with glycerol, andcompared to sodium hyaluronate（SH）, exploring the effect of penetratingkeratoplasty using corneas preserved by different conditions so as to provideexperimental evidence for the useful of corneal preservation and cornealtransplantation in clinic.MethodsEyes of40female Wistar rats with80of the corneal pieces were dividedinto four groups randomly by random number method: normal controls,chondroitin sulfate group（Ⅰ）, sodium hyaluronate group（Ⅱ）, pure glyceringroup（Ⅲ）. The normal control group of fresh corneals were not stored, butconducted the further experiments directly. TheⅠ、Ⅱ group were evenlyanointed on the endothelial surface of the cornea by1%CS and1%SH thencryopreserved in glycerol, while the Ⅲ group were cryopreserved in glycerindirectly.2months later, all the preserved group was examined viability withtrypan blue combined with alizarin red staining for corneal endothelial cells,while electron-microscope examination for observing the changes of theultrastructure, and all the preserved corneal was received the allogeneic penetrating keratoplasty（PKP）. The rejection index（RI）and the means survivaltime（MST）on each group of corneal grafts were recorded by slit-lampmicroscope. Hematoxylin and eosin staining was used to reflect the infiltrationdegree of the neovascularity and the lymphocytes on corneal grafts aftercorneal transplantation. And the immunohistochemistry were adopted toexamine the expressions level of TGF-β1and ICAM-1in corneal grafts so as tofurther test the effect of transplantation for each groups.ResultsCECs morphology was normal in fresh cornea in normal control groupwithout blue stained cells. In CS group, few blue-stained cells were seen,however, the numbers of blue-stained cells were obviously increased in SHgroup and only glycerin group. The viability and density of CECs weresignificantly lower in SH group and only glycerin group compared with CS group（P＜0.05）. The edema of the endoplasmic reticulum were severer in SHgroup and only glycerin group than the CS group on2months afterpreservation. The neovascularity and the lymphocytes infiltration were severerin SH group and only glycerin group than the CS group on14days after PKP.Compared with SH group and only glycerin group, the RI in CS group wassignificantly reduced and the MST was delayed after PKP （P＜0.05）.Immunochemistry revealed that the expression of TGF-β1in grafts wasupregulated, and that of ICAM-1was downregulated in CS group comparedwith SH group and only glycerin group（P＜0.05）. Meanwhile, CS group ofgrafts approached to the expression level in normal control group.ConclusionsIn cryopreservation of cornea, CS could protect and stabilize cornealendothelial cells. CS could obviously improve the effect of cryopreservation withglycerol, applied in the models of animal penetrating keratoplasty can gainsatisfactory postoperative effects which surpass SH. CS used for the long-term cornea preservation, as this could provide better donor for clinical cornealtransplantation.