Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone.
An ultrastructural and immunohistochemical comparison was made between the effects of Dispase II and EDTA on the basement membrane zone of corneal epithelium. The comparison was made on intact corneas as well as on freed epithelial sheets and remnant stromas that had been separated using either the enzyme or chelator. At the ultrastructural level, incubation with Dispase II disrupted the lamina densa allowing in situ blebbing of the basal cells. After separation of the epithelium and stroma, the epithelial sheets showed extensive blebs with extracellular matrix trapped between the blebs. The remnant stromas completely lacked lamina densa, but anchoring fibrils remained. Immunofluorescent studies with antibodies against laminin and BM-1 antigen (an antibody to the protein core of heparan sulfate proteoglycan) revealed that laminin antibody binding was present on the freed epithelial sheets and absent from the remnant stromas, whereas BM-1 antibody binding was absent from both the freed sheets and the remnant stromas after incubation with Dispase II. Incubation with EDTA did not disrupt the basal lamina. There was no in situ blebbing of the basal cells, but immediately after epithelial sheet removal, extensive blebbing occurred. Freed sheets lacked attached segments of extracellular matrix as seen with Dispase II treatment. Remnant stromas showed intact basal laminae and anchoring fibrils. Immunofluorescent studies revealed binding of both the laminin and BM-1 antibodies to the remnant stromas, but not to the freed epithelial sheets. Although EDTA removal of epithelial sheets gave a cleaner separation between basal cell membranes and the basal laminae, more basal cells were disrupted than after separation with Dispase II.
D000818:Animals; D001485:Basement Membrane / Q000187:drug effects / Q000648:ultrastructure; D003315:Cornea / Q000187:drug effects / Q000648:ultrastructure*; D004492:Edetic Acid / Q000494:pharmacology*; D010450:Endopeptidases / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D004492:Edetic Acid; D010450:Endopeptidases; C023139:dispase