SURFACTANT-SKIN INTERACTIONS 2 31 Figure 7. Representative ESEM pictures of the surface of tissues treated with water (a), formulation A (b), formulation B (c), and formulation C (d). Arrows show the uplift of the individual corneocytes. the disjunctum were occasionally encountered. Such regions displayed multilayers of intact sheets of lipid lamellae around and between neighboring desmosomes. Similar features have also been reported in sequential tape strips harvested from normal skin, which revealed that the multiple lamellae existed only in the lower layers of stratum corneum and were absent toward the surface layers (2). Formulation A-treated skin was similar in morphology to water-washed tissue except that regions devoid of disjuncture appeared with increased frequency. Intracellular proteins and extracellular lipids remained predominantly unaffected. Lipid lamellae were multilayered and well-formed. The water loss did not significantly depend on the damage to the outer cell layers (which lack lipid lamellae), presumably because of two reasons: first, the damage to outer layers was common to both water and formulation A-treated pieces of skin, and second, the TEWL values from different tissue pieces were fairly consistent. The general structure of formulation B-treated tissue was similar to formulation A-treated tissue except for significant inter- and intracellular damage to about 25% of the tissue noticed in the case of formulation B. Lipids often lacked characteristic periodicity in formulation B-treated tissue. TEWL values were modest. Regional variation in TEWL could be attributed to the damaged lipid lamallae observed in about 25% of the tissue. Formulation C-induced damage was ubiquitous and severe, and resulted in loss of lipid structure as well as intracellular damage to proteins. Such

232 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS damage was reflected in high TEWL values. The nature of structural damage was similar in all specimens irrespective of individual variations in TEWL. The fact that only the upper layers appeared damaged in tissue displaying lower TEWL numbers (11 g/m 2 hr) in contrast to four to six affected layers for high TEWL numbers (35 g/m 2 hr) indicates a direct relationship between the extent of damage and water loss. TEWL is generally considered to be a passive diffusion phenomenon. Assuming Fick's law of diffusion as a first approximation, diffusion of water could be considered inversely proportional to the distance or the total thickness of the corneum. However, water loss through the surface layers of corneocytes is not only inversely proportional to the thickness of the epidermis but also depends upon the resistance to its migration pathway provided by the barrier lipids. Both parameters (epidermal thickness and lipids) were affected by different surfactant treatments. Although the epidermis often displayed structural variability even in water-treated tissue, the decrease in corneum thickness was not significant enough to affect TEWL, as was evident by the uniformity of TEWL measurements. Also, in water-treated tissue the lipids were well preserved in the lower layers of the stratum corneum, providing an effective barrier against water loss. Treat- ment with the isethionate bar resulted in an increase in the frequency of occurrence of intact multilamellar lipid sheets covering the upper corneocyte layers. This suggests that the treatment with formulation A resulted in a reduction (compared to control) in the outer layers of disjunctum and consequently caused a slight increase in TEWL. The water loss, however, remained limited due to the barrier offered by the surface- contouring lameliar lipids. Glycerin bar-treated tissue displayed features similar to those described for the isethionate bar. However, the outer lipids displayed damaged mor- phology, i.e., non-lamellar, amorphous material in about 25% of the surface regions. It is tempting to suggest that the modest increase in TEWL in glycerin bar-treated tissue could be ascribed to damaged lipids in the outer layers of surface corneocytes, but the relationship is only directional at best. Significant dependence of TEWL on the thinning of epidermis and disrupted lipid structures was evident in soap bar-treated tissue. The disjunctum was compromised in almost 90% of the tissue. The lipid barrier underlying the disjunctum was severely affected and appeared as an amorphous non-lamellar struc- ture. In tissue displaying 30 g/m 2 hr of water loss, the stratum corneum was punc- tuated with regions in which almost all structure above the viable cell layers was disrupted (Fig. 6c). The soap bar-treated tissue revealed a decrease in the number of cell layers forming the stratum corneum, lipid loss, and significant swelling of the corneocytes. Some of the corneocytes were four times wider in projection compared to the normal corneocytes. A surfactant-induced increase in surface area and swelling is known to depend on the surfactant type (13). Increased permeability of the cell membrane and consequent in- crease in the intracellular binding of water molecules may be responsible for the observed increase in the projected width of damaged corneocytes. An advantage of using ESEM for structural studies is that the specimen can be observed in its native state without preparation artifacts. However, the interpretation of the ESEM micrographs requires a careful analysis. Normal, undamaged skin in ESEM appears as a smooth surface. In damaged skin, the structural changes to corneocytes appear as brighter regions. This could be attributed to the uplifting of cells, which causes changes in their geometric disposition. Peripheries of uplifted corneocytes appear brighter than surrounding regions. Such fine changes are often difficult to detect because the skin