| Objectives1. To make up organ culture mediums containing fetal bovine serum (FBS) and human umbilical cord serum (HUCS). Porcine corneas were organ cultured with these two mediums. To evaluate the changes of corneal endothelial morphology, histology, ultrastructure, enzymes activity and metabolism induced by corneal preservation in these two mediums.2. Penetrating keratoplasty was performed on New Zealand rabbits. The donors were organ cultured for 4 weeks in the two mediums. The outcome was evaluated by corneal transparency, endothelial cell density, graft thickness and histology postoperation.3. Human corneas were organ cultured in the two mediums. To observe corneal endothelial morphology, histology, ultrastructure and metabolism. Proliferative activity and HLA-DR antigen of corneas were also observed after organ culture.Methods1. Make up organ culture medium, medium I containing 10% FBS and medium II containing 10% HUCS. Fifty pairs of porcine corneas were preserved at 3IX? for 7, 14, 21, 28 days. One cornea of each pair was cultivated in medium I (group 1); the other onewas stored in medium II (group 2). Thirteen fresh porcine corneas served as controls. The medium was not changed during culture. All stored corneas were deswollen for 24 hours by addition of 5% dextran T500. At weekly intervals, 12 corneas from each group was evaluated. Twelve fresh porcine corneas served as controls. The endothelium was examined by induced dilation of intercellular spaces with inverted light microscopy. Then the corneas were stained with trypan blue and alizarin red and endothlial was examined by light microscopy. Six of the 12 corneas were examined for histological study. The enzyme activity of other 6 corneas was evaluated by enzyme histochemical staining of lactate dehydrogenase, succinic dehydrogenase, adenosine triphosphatase. Scanning electron microscopy was performed on one cornea from each group at 14 and 28 days and one fresh cornea. PH value, glucose and lactate concentration of medium before and after culture were examined. Samples for microbiological evaluation were obtained from eyeball cotton swab, storage medium and deswelling medium.2. Forty-five New Zealand rabbits were divided into three groups. Ten rabbits of group 1 were performed penetrating keratoplasty with donor corneas kept for 4 weeks in medium I. Ten rabbits of group 2 were grafted with paired donor corneas kept for 4 weeks in medium II. Ten rabbits of group 3 were grafted with fresh corneas which endothelium was scraped. Corneal transparency was evaluated by slit-limp microscopy. At 3, 7, 14 days, grafts endothelium was examined by specular microscopy and grafts thickness were examined by pachymetry. At 7, 14 days, 5 grafts ofeach group were cut off and stained with trypan blue and alizarin red. Histology was also studied. Microbiological evaluation was performed during organ culture.3. Three pairs of human corneas were organ cultured at 31癈. One cornea of each pair was cultivated in medium I (group 1); the other one was stored in medium II (group 2). One pair was examined endothelium at 21, 28 days. Corneas were then divided into two pieces for histological study and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and HLA-DR antigen. Scanning electron microscopy and transmission electron microscopy (TEM) were performed on one pair at 28 days. PH value, glucose and lactate concentration of medium before and after culture were examined. Microbiological evaluation was performed during organ culture.Results1. Endothelial evaluation did not differ statistically between the two groups of porcine corneas. The endothelial morphological study showed some alterations such as pleomorphism. After 28 days of cultivation, a mean cell loss of 10.98% was found in medium I stored corneas and 10.85% in medium II stored corneas. There are no statistical differences for the histology, ultr... |
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