222 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). After extensive washing, tissue pieces were post-fixed in either buffered 1% osmium tetroxide (30 min) or 0.5% ruthenium tetroxide (15 min). Samples were rinsed in three changes of buffer before dehydration in a graded concentration series of acetone. Specimens were embed- ded in Spurr resin and incubated at 60øC for two days. Ultrathin sections were cut from the hardened blocks, mounted on carbon-coated copper grids, and post-stained with uranyl acetate and lead citrate. The specimens were examined in a JEOL 1200 EX TEM. RESULTS TEWL TEWL measurements were obtained at eight sites for each of the treatments, and specimens were divided for further structural studies using TEM and ESEM (Table II). The distribution of TEWL measurements was narrow for tissue exposed to water, formulation A, and formulation B. The respective mean (+SD) values of TEWL were 0.78 + 0.4 g/m2hr (water), 2.3 + 0.8 g/m2hr (formulation A), and 3.3 + 0.9 g/m2hr (formulation B). On the other hand, tissue treated with formulation C exhibited the highest TEWL values and showed significant site-to-site variation (24 + 16 g/m2hr). TEM AND ESEM Water-washed control skin. Epidermis, in a water-washed control specimen (Figure la), consists of the inner and outer layers of stratum corneum (compacturn and disjunctum). Disjuncture is characterized by the absence of interlayer desmosomes. In contrast, the corneocytes in successive layers of compactum exhibit the presence of interlayer desmo- somes. The status of desmosomes may differ depending primarily upon the extent of degradation caused by desquamating enzymes. The intercellular space is generally oc- cupied by sheets of lipid lamellae (12), the number and integrity of which vary from region to region. The outermost layers of the disjunctum normally displayed a thin sheet of lipids comprising one to two layers (Figure lb). Departures from this normal outer stratum corneum structure were occasionally observed in those regions of water-washed tissue where stratum corneum appeared either totally or partly devoid of disjunctum (Figure lc). The outer lipid layers, in such regions, displayed robust structures of well-formed multilayers (five or six) that extended around and between desmosomes (Figure ld). In general, the number of lipid lamellae were larger in the lower layers of corneocytes, which also displayed a greater number of interlayer lipid-enveloped des- mosomes. A majority of the tissue, however, displayed intact disjunctum, with very few lipid layers covering the outer corneocytes. Multilamellar lipid layers were seen envel- oping only 20-30% of the surface corneocytes. Structurally, the intercellular proteins and cell envelope appeared normal and remained unaffected by treatment. TEWL values for water-washed tissue pieces did not show significant site-to-site variation. This is confirmed by the uniformity of the structural preservation observed in different pieces of tissue. Even in the 20-30% of the region that displayed damaged disjunctum, the intact lipid multilayers were presumably sufficient to maintain barrier properties. ESEM showed that the surface topography was fairly smooth (Figure 7a), the individual cor-

SURFACTANT-SKIN INTERACTIONS 223 neocytes were hardly visible, and very few corneocytes displayed noticeable uplifting (uplifted corneocytes appear brighter with greater contrast). Formulation A-treated skin. The overall ultrastructural morphology of skin exposed to the isethionate bar (formulation A) appeared very similar to that of the water-washed skin. However, in general, fewer cell layers were present in the disjunctum/compactum re- gions (Figure 2a). Thin lamellar sheets or multilayer lipid regions were seen in both formulation A- and water-treated skin. The lipid lamellae in the outer layers were often present as uniform, 4-8 layers thick, well-defined extended regions (Figure 2b) and, as in the water-washed specimens, these regions belonged to the lower layers of stratum comeurn, often in compacturn. The integrity of proteins within the cell and the cell envelope was noticeably unaffected by the treatment. Formulation A-treated skin ex- hibited somewhat higher TEWL values than water-washed skin. This difference may be due to an overall reduction in the number of cell layers and a resultant decrease in the thickness of the corneum. Corneocyte size was unaffected. The surface topography (Fig- ure 7b) was smooth, with a few uplifted corneocytes visible. Formulation B-treated tissue. The glycerin bar (formulation B)-treated specimen was simi- lar to the skin specimen treated with the isethionate bar. The number of cell layers in the disjunctum/compactum region was variable. Although the majority of the tissue displayed the normal morphology of lipid lamallae, evidence of disorder within the lamallae could occasionally be seen (Figure 3a,b). Generally, the organization of cell envelope and proteins was unaltered. In certain cases, however, the tissue displayed significant disruption of proteins and lipids (Figure 4a,b). The intercellular lipids in adjacent outer layers were either absent or displayed disordered structures. Damage to cellular regions was evident in the form of large, globular, low-density regions, which often displayed lipid-like structures (arrows, Figure 4b). Such damage was always con- fined to the outermost layer of the epidermis. Formulation B-washed tissue appeared somewhat more damaged in comparison to either control or to formulation A-treated tissue. This was reflected in terms of somewhat larger TEWL values. ESEM observations also showed the presence of a greater number of uplifted corneocytes compared to control (Figure 7c). Formulation C-treated tissue. The soap bar (formulation C)-treated specimen exhibited maximum ultrastructural damage to cellular proteinaceous material, envelope, and la- meliar lipid structures. The presence of disjunctum could be seen only in about 5% of the treated tissue (Figure 5a). Most of the tissue displayed absence ofdisjunctum and loss of a few layers of compacturn. Lipid structure in the outer layers was often severely damaged. Ordered lamellar structures were replaced by amorphous material. Such re- gions often coexisted with poorly ordered lipid-like structures (arrows, Figure 5b). This could be due to the intercalation of detergent in lipid layers. Intracellular damage to proteins was similar to that described for formulation B except that the damage was much more extensive and severe in the formulation C-washed tissue (almost 25% of the formulation B-washed tissue and 100% of the formulation C-washed tissue displayed intracellular damage). The damaged cells appeared three to four times wider than the control cells (Figures 6a, lc). TEWL measurements showed significant site-to-site variation. The lowest TEWL value for the skin examined using TEM was 11 g/m 2 hr. Damage in this case was confined to the top two layers of corneocytes. Specimens that showed large water loss (-35 g/m 2 hr)