The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods. A scanning and transmission electron microscopical study.
The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads". Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t
Glasø
M
M
Electron Microscopical Laboratory, University of Oslo, Norway.
Håskjold
E
E
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003470:Culture Media; D000078404:Epidermal Cells; D004817:Epidermis / Q000648:ultrastructure*; D005260:Female; D008297:Male; D051379:Mice; D008812:Mice, Hairless; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D003470:Culture Media; D014357:Trypsin; D002118:Calcium