DIMETHYL SULFOXIDE AND SKIN 131 Methods Sheets of stratum corneum which were conditioned for 24 hours at 21øC and RH 65 ñ 2% were cut into strips (11/4 in. X 1/4 in.) and at- tached to small tabs by means of an epoxy resin. Only strips without breaks, tears, or obvious holes were considered suitable for the experi- ments. The strips of stratum corneum were then immersed for about 24 hours in CHCla-saturated water (this medium was used in order to pre- vent microbiological decomposition), and calibrated by stretching them to approximately 5% extension in distilled water on the Instron Exten- someter operating at a constant rate of extension (0.1 in./min). Cell A with the high sensitivity setting which gave a fall scale load of 2 g was used in most cases. The strips were relaxed overnight in CHCla-saturated water and then transferred for an additional 24 hours into the solvent in which they were restretched. It was not determined whether or not this 24-hour sol- vent incubation time was sufficient for equilibrium to be reached. In order to eliminate the differences between various samples, the same skin sample served as its own control and test. As many experiments as pos- sible were done on the skin of one animal, especially when the effect of the solvent concentration on the work index was examined. It is relatively difficult to obtain a large and uniform piece of guinea pig stratum comeurn and, therefore, it is not practically possible to cut all the strips in the same direction with the ridges and grooves running perpendicular or parallel to the applied force. In order to avoid any artifacts resulting from different orientations of the strips, the direction of the striations was noted before the stratum comeurn strips were tested on the Instron. The results indicated that there was no significant dif- ference between strips cut with the striations perpendicular or parallel to the applied force. The changes in mechanical properties were evaluated by calculating the ratio of the work needed to stretch the skin sample approximately 5% in a given solvent (or a repeat time in water) to that measured during its initial calibration in water (15, 16). Work index = work required to stretch strip 5% in the solvent (or a repeat time in water) work required to stretch strip 5% in water ini,tially In order to calculate the work necessary to stretch the tissues, the

132 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS work profiles were cut from the Instron charts and weighed. Thus, the work index was calculated from the ratios of the weights before and after treatment. The reversibility of the solvent effect was assessed by rinsing the tested strips free of solvent with distilled water, relaxing them overnight in CHCla-saturated H20, and restretching them a third time in water. The work obtained was compared to that observed during the calibra- tion and expressed as the work index. Slight modifications of the testing procedure were made in the follow- ing cases: (a) During experiments with ammonium thioglycolate the strips were calibrated in water and relaxed in water as usual. Then they were trans- ferred for approximately 15 minutes to the appropriate reagent (am- monium thioglycolate or ammonium chloride) and restretched in dis- tilled water. (b) In the case of 97% formic acid, the strips were calibrated and re- laxed in water as usual, and then transferred to the reagent for 2 minutes and restretched in distilled water. RESULTS AND DISCUSSION Relaxation of Stratum Corneum Strips after Stretching in Water In a first series of experiments, the reversibility of the stress-strain characteristics of human and guinea pig stratum corneum as a function of per cent extension was examined. The strips which were prepared in the usual manner were stretched to 5, 10, and 20% extension in distilled water, relaxed in C HCla-saturated water, and restretched in water (Table I). The human strip stretched for 20% extension seemed to be irreversibly damaged and the repeat load-extension curve showed a marked difference from the untreated one. Guinea pig strips stretched for 20% extension (not reported) also appeared irreversibly changed. Those human and guinea pig strips stretched up to 10% extension in water relaxed completely and gave identical curves even after immedi- ate recycling. Guinea pig strips were stretched 5% for three times in water follow- ing a stretch-relax-stretch-relax-stretch procedure. The differences in work index which were observed (Table II) were not considered to be significant. Other experiments (not reported) indicated that five re-