Imbert D, Cullander C
Department of Biopharmaceutical Sciences, School of Pharmacy, University of California at San Francisco 94143-0446, USA.
Cornea. 1997 Nov;16(6):666-74.
Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness.
New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay.
Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only.
This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.
确定切除角膜的活力不仅对移植储存评估有意义，对体外扩散研究设计和眼毒性评估也很重要。通过分别使用钙黄绿素 AM（CAM）和碘化丙啶同型二聚体 -1（EH -1）进行同步活细胞染色作为“活”和“死”探针，我们开发了一种共聚焦激光扫描显微镜（CLSM）检测方法来确定上皮和内皮的活力并估计角膜厚度。
将新西兰白兔角膜储存在 4℃或室温的磷酸盐缓冲盐水（PBS）或Optisol中。在不同时间，用EH -1/CAM溶液对角膜进行染色，不经进一步处理，通过CLSM观察。还使用MTT检测评估储存对角膜的影响。
在室温下于PBS中储存4小时后，观察到基质肿胀、上皮上层脱落和严重的内皮损伤。8小时后，观察到下层上皮细胞死亡以及内皮结构丧失。在Optisol中于类似条件下储存的角膜与对照无差异。在4℃下于Optisol中储存7天和14天时，对角膜上皮表层的影响相似。在25天时观察到广泛的上皮脱落和翼状细胞死亡，但基底层仍约50%保持健康。在25天时观察到明显的内皮细胞损失。MTT结果仅在中期储存研究中与CLSM数据一致。
这种CAM/EH -1 CLSM荧光检测方法是角膜活力的敏感指标，因此在不同细胞层维持重要功能至关重要的研究中可能证明是有用的。
