Nelson L R, Hodge D O, Bourne W M
Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Cornea. 2000 Nov;19(6):782-7. doi: 10.1097/00003226-200011000-00005.
To compare paired human corneas after storage at 4 degrees C in Chen medium (CM) and Optisol-GS medium (OM) for 7, 10, 14, and 21 days.
One cornea of each pair from nine human donors was randomly stored in either CM or OM, with its mate cornea stored in the other medium. Three pairs of corneas were stored for 7 days and two pairs each were stored for 10, 14, and 21 days at 4 degrees C. Baseline corneal thickness measurements and endothelial photographs were obtained with a specular microscope. Corneal thickness measurements were also taken on days 7, 10, 14, and 21 of storage. At the end of storage, the corneas were warmed 2 hours before endothelial photographs were taken and were then placed in fixative. A corneal endothelial analysis system was used to compare changes in endothelial size and shape after storage. After fixation, the corneal endothelium was examined by scanning electron microscopy (SEM), and TdT-dUTP terminal nick-end labeling (TUNEL) assays with 4'6-diamidino-2-phenylindole (DAPI) counterstaining were performed on tissue sections of each cornea. A laser scanning confocal microscope and an automated digital analysis system were used to detect the presence of TUNEL-positive apoptotic cells in each cell layer and to determine keratocyte densities.
Mean corneal thickness at 0, 7, 10, 14, 21 days of storage was 0.69 +/- 0.05 mm, 0.69 +/- 0.06 mm, 0.73 +/- 0.08 mm, 0.87 +/- 0.04 mm, and 0.87 +/- 0.03 mm, respectively, for CM and 0.65 +/- 0.06 mm, 0.59 +/- 0.07 mm, 0.63 +/- 0.03 mm, 0.60 +/- 0.03 mm, and 0.69 +/- 0.02 mm, respectively, for OM (p < 0.0001). The mean decrease in endothelial cell density at the end of the 7-, 10-, and 14-day storage periods was 11 +/- 10% for the CM corneas and 5 +/- 5% for the OM corneas (p = 0.18). SEM showed an intact endothelial monolayer in all corneas. The mean percentages of TUNEL-positive cells in epithelium, stroma, and endothelium of CM-stored corneas were 4 +/- 4%, 2 +/- 3%, and 0.1 +/- 0.3%, respectively, and did not differ from the OM-stored corneal values of 4 +/- 3%, 2 +/- 4%, and 0.9 +/- 1.5%. The percentage of TUNEL-positive cells did not increase with storage time. Keratocyte density was 368 +/- 130 cells/mm2 for CM-stored corneas and 447 +/- 96 cells/mm2 for OM-stored corneas (p = 0.13).
Corneas stored in CM were thicker during storage than those stored in OM. The two storage media did not differ with respect to endothelial cell loss during storage or to the percentage of TUNEL-positive cells or keratocyte density at the end of the storage period.
比较在4℃下于陈培养基（CM）和Optisol - GS培养基（OM）中储存7、10、14和21天的配对人角膜。
来自9名人类供体的每对角膜中的一只随机储存在CM或OM中，其配对角膜储存在另一种培养基中。三对角膜在4℃下储存7天，两对角膜分别储存10天、14天和21天。使用镜面显微镜获得基线角膜厚度测量值和内皮细胞照片。在储存的第7、10、14和21天也进行角膜厚度测量。储存结束时，在拍摄内皮细胞照片前2小时将角膜复温，然后放入固定剂中。使用角膜内皮分析系统比较储存后内皮细胞大小和形状的变化。固定后，通过扫描电子显微镜（SEM）检查角膜内皮，并对每个角膜的组织切片进行4',6 - 二脒基 - 2 - 苯基吲哚（DAPI）复染的TdT - dUTP末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）分析。使用激光扫描共聚焦显微镜和自动数字分析系统检测每个细胞层中TUNEL阳性凋亡细胞的存在并确定角膜细胞密度。
对于CM，储存0、7、10、14、21天时的平均角膜厚度分别为0.69±0.05mm、0.69±0.06mm、0.73±0.08mm、0.87±0.04mm和0.87±0.03mm；对于OM，分别为0.65±0.06mm、0.59±0.07mm、0.63±0.03mm、0.60±0.03mm和0.6９±0.02mm（p<0.0001）。在7天、10天和14天储存期结束时，CM储存的角膜内皮细胞密度平均降低11±10%，OM储存的角膜内皮细胞密度平均降低5±5%（p = 0.18）。SEM显示所有角膜中的内皮单层完整。CM储存角膜的上皮、基质和内皮中TUNEL阳性细胞的平均百分比分别为4±4%、2±3%和0.1±0.3%，与OM储存角膜的4±3%、2±4%和0.9±1.5%的值无差异。TUNEL阳性细胞的百分比未随储存时间增加。CM储存角膜的角膜细胞密度为368±13０个细胞/mm²，OM储存角膜的角膜细胞密度为447±96个细胞/mm²（p = 0.13）。
储存在CM中的角膜在储存期间比储存在OM中的角膜更厚。两种储存培养基在储存期间内皮细胞损失、储存期结束时TUNEL阳性细胞百分比或角膜细胞密度方面无差异。
