Fluorescence confocal laser scanning microscopy for in vivo imaging of epidermal reactions to two experimental irritants

Background: Fibre-optic fluorescence confocal laser scanning microscopy (CLSM) is a novel non-invasive technique for in vivo imaging of skin. The cellular structure of the epidermis can be studied. A fluorophore, e.g. fluorescein sodium, is introduced by an intradermal injection or applied to the skin surface before scanning. Images are horizontal optical sections parallel to the skin surface. Fluorescence CLSM has hitherto not been applied to experimental contact dermatitis. Objective: The aim was to study the applicability of fluorescence CLSM for in situ imaging of irritant contact dermatitis reactions caused by established model irritants, e.g. sodium lauryl sulphate (SLS) and pelargonic acid (PA). Methods: Twelve healthy individuals volunteered. The flexor aspect of the right and the left forearm was exposed to SLS in water and PA in isopropanol and occluded under Finn Chambers for 24 h. The reactions were rated clinically and, following epicutaneous and intra-dermal application of fluorescein sodium, studied by fluorescence CLSM, magnification x 1000. Results: Both irritants disturbed the epidermal intercellular borders, which became blurred, thickened and variably altered. This was interpreted as being a result of chemical damage to cellular membranes. Cell borders might show a double contour as a result of inter-cellular oedema. PA might increase the size of individual keratinocytes interpreted as a result of intra-cellular disturbance with oedema. SLS-exposed sites showed clusters of keratinocytes with visible nuclei in the outer layers of the epidermis, e.g. a parakeratotic shift supposed to be due to increased cell proliferation elicited by SLS. The isopropanol vehicle and PA did not interfere with the CLSM imaging technique or the experimental procedures. SLS, being a detergent, however, modified the physico-chemical properties of the skin surface and both disturbed epicutaneous labelling with the flurophore and immersion oil coupling between the skin surface and the optical system. Thus, SLS was technically more difficult to study by CLSM than PA. Conclusions: This preliminary study demonstrated the applicability of fluorescence CLSM for a detailed study of experimental skin irritants in vivo. Essential findings were disturbed and widened cell borders, swelling of keratinocytes by PA and induction of a parakeratotic shift by SLS with clusters of keratinocytes holding nuclei in the epidermis. Fluorescence CLSM offers a unique opportunity to study the inter- and intracellular water compartments directly in the epidermis in situ and an opportunity to visualize cell proliferation manifested as parakeratosis. Fibre-optic fluorescence CLSM of irritant reactions is, however, technically more complicated than reflectance CLSM and may not be applicable to any irritant. SLS applied epicutaneously interacted with the skin surface and coupling to the microscope and was thus found to be more difficult to study technically than PA. PA dissolved in isopropanol is for technical reasons, and with SLS as alternative, considered the preferred model irritant.