An experimental study on the combination of chondroitin sulfate with glycerin for the cryopreservation of corneal graft

Abstract

Background Chondroitin sulfate(CS) is a highly viscous and elastic acid mucopolysaccharide extracted from animal soft tissues,with a wide range of biological activity for use in clinical ophthalmology.Interim preservation solution containing CS has a significant protective effect on corneal endothelial cells (CECs).However,the protective effect played by CS in long-term glycerol cryopreservation of CECs remains to be studied. Objective This study is mainly attempted to investigate the protective effect of CS on graft CECs after cryopreservation by glycerin,and to compare the preserving outcome with that of sodium hyaluronate (SH). Methods One hundred and four eyes of fifty-two female Wistar rats were divided into four groups randomly.The cornea grafts were evenly anointed on the surface of the endothelium by 3％ CS and 1％ SH,respectively,and then cryopreserved in glycerol in the 3％ CS group and 1％ SH group,and the corneas cryopreserved only in glycerin were assigned to the glycerin only group.The fresh corneas of matched rats were used as the normal control group.Ninety-six female SD rats were appointed as recipients to receive allogeneic penetrating keratoplasty (PKP) 2 months after the graft cryopreservation.The viability of CECs on the corneas cryopreserved for 20 days and grafts of 14 days following surgery was assessed using trypan blue & alizarin red stain.The ultrastructure of cryopreserved corneas was examined under a transmission electron microscope.The rejection index (RI) and graft survival time were evaluated by inflammatory scoring under the slit lamp 1 day after operation,and regular pathological examination was performed after 7,14 or 30 days.The expression of transforming growth factor-β1 ( TGF-β1 ) and intercellular adhesion molecule-1 ( ICAM-1 ) in corneas and grafts were detected by immunochemistry. Results CECs morphology was normal in fresh cornea in normal control group,presenting no blue stained cells after trypan blue staining.In the 3％ CS group,few blue-stained cells were seen:however,the number of blue-stained cells noticeably increased in the 1％ SH group and glycerin only group.The viability and density of CECs were significantly lower in the 1％ SH group and glycerin only group,compared with the 3％ CS group (P＜0.05).Edema of the endoplasmic reticulum,inflammatory infiltration and neovascularization were more severe in the 1％ SH group and glycerin only group than the 3％ CS group 14 days after PKP.Compared with the 1％ SH group and glycerin only group,the R1 of the 3％ CS group was significantly reduced and the CECs survival time was delayed after PKP ( P＜0.05 ).Immunochemistry revealed that the expression of TGF-β1 in grafts (A value)was up-regulated and that of ICAM-1 was down-regulated in the 3％ CS group,compared with the 1％ SH group and glycerin only group ( P＜0.05). Conclusions The combination of 3％ CS with glycerol to cryopreserve corneal donor can maintain the viability of CECs for a longer duration and improve the effectiveness of PKP. Key words: Chondroitin sulfate; Penetrating keratoplasty; Graft cryopreservation; Corneal endothelial cell; Glycerin; Sodium hyaluronate