Diffusion of [2-14C]diazepam across isolated hairless mouse stratum corneum/epidermal tissues

The objective of this study was to develop a method of preparing mouse stratum corneum/epidermal (SCE) tissue without the dermis for use in drug diffusion studies. The diffusion of radiolabeled diazepam across this new preparation has been studied and the effect of the dermis on diffusion evaluated. Incubation of large pieces of mouse skin in a 20mM EDTA, 15 mM sodium phosphate buffer, pH 7.2, in normal saline for 3-4 h at 37 degrees C resulted in a tissue which easily separated at the epidermal-dermal junction. The resulting tissue contains stratum corneum and epidermis, which are the same layers used in studies with human skin in vitro. The EDTA treatment did not effect diffusion of [2-14C]diazepam across whole mouse skin (SCE and dermis) used as controls. The rate of drug diffusion was greater across SCE than SCE and dermis, however, 0.48-1.12 micrograms/cm2/h versus 0.11-0.52 microgram/cm/h, respectively. The permeability coefficients for mouse SCE ranged from 1.92-4.48 X 10(-2) cm/h. The lag times and diffusion coefficients were 0.36-0.91 h and 0.1-0.6 X 10(-6) cm2/h, respectively. The presence of the dermis decreased the diffusion rate or flux of diazepam. The dermis appears to accumulate drug until it is saturated and then the drug diffuses into the receiving chamber.     

Determination and correlation of in vitro viability for hairless mouse and human neonatal whole skin and stratum corneum/epidermis

Background and design: Viable tissue is essential to assess the rate and extent of biotransformation during percutaneous absorption in vitro. We assessed the viability of hairless mouse whole skin (WS) and stratum corneum/epidermis (SCE) and human neonatal SCE following separation from the dermis by EDTA phosphate-buffered saline (EDTA-PBS) incubation or by heat treatment by measuring the conversion of dextrose to lactate. Lactate concentrations in receptor fluid samples were determined using a Sigma diagnostic lactate determination kit. A standard curve was prepared and samples assayed spectrophotometrically at 340 nm using a lambda 2β spectrophotometer. Standard curves were prepared for each experiment and correlation coefficient values ( r ) were calculated. Results: Our results showed that heirless mouse SCE was associated with glucose conversion to lactic acid at an increased rate if incubated in EDTA-PBS for 4 h and used immediately. Lactate production was greater with the dermis present (EDTA-PBS WS). The rate of glucose to lactate conversion in hairless mouse SCE was 20–25% of that found in WS. Compared with Dulbecco’s modified PBS (DMPBS)-treated WS controls, the rate of lactate production in EDTA-PBS-treated WS was nearly a 50% less. Heat treatment in water at 60° C to separate SCE from hairless mouse WS appeared to eliminate viability. Viability of hairless mouse SCE, as measured by glucose conversion to lactate, was comparable to human neonatal SCE. Conclusions: These results suggest that the dermis is a significant contributor to glucose metabolism and that incubation in EDTA-PBS is a contributing factor to the overall decrease in metabolic capacity of the tissue. As a result of these findings, hairless mouse SCE appears to be useful as a model for human neonatal SCE in percutaneous absorption studies. Received: 1 September 1995     

Effect of lactic acid isomers on keratinocyte ceramide synthesis,stratum corneum lipid levels and stratum corneum barrier function

Alpha-hydroxy acids are effective agents for the treatment of skin xerosis and it is known that, following treatment with lotions containingd,l-lactic acid, the stratum corneum prevents xerosis more effectively. To date, the relative efficacy of the different isomers of lactic acid has not been evaluated and the mode of action of lactic acid in improving stratum corneum resilience is not known. The objective of the present studies was to determine the effects of lactic acid isomers on keratinocyte ceramide biosynthesis, stratum corneum barrier function and the resistance of the straum corneum to the appearance of skin xerosis. In vitro, lactic acid enhanced the production of ceramides by keratinocytes.l-Lactic acid was more effective than thed isomer (300% increase vs 100% increase). Carbon label from lactic acid was incorporated into all keratinocyte lipid species and a greater incorporation of label into ceramides was achieved withl-lactate than withd-lactate. In vivo, lactic acid increased the levels of stratum corneum ceramides. Whereas, lotions containingl-lactic acid resulted in the greatest increase (48% increase) followed byd,l-lactic acid (25% increase),d-lactic acid had no effect on stratum corneum ceramide levels. The increases in stratum corneum ceramide levels following lactic acid treatment also led to improvements in stratum corneum barrier function, measured by transepidermal water loss following a challenge to the skin with SLS and in the regression phase of a moisturization efficacy study. Significant improvements in barrier function and resistance to the appearance of skin xerosis were observed followingl-lactic acid andd,l-lactic acid, but not followingd-lactic acid treatment. From these results we believe that lactic acid, particularly thel isomer, stimulates ceramide biosynthesis leading to increased stratum corneum ceramide levels which results in superior lipid barrier and a more effective resistance against xerosis.     

Properties of acid phosphatase in human stratum corneum

Sheets of stratum corneum were prepared by a trypsinization procedure from human skin samples, homogenized with a freeze press and then fractionated into a soluble fraction and a sediment by centrifugation at 50,000 g. Acid phosphatase (AcP) activity was found in both fractions but the bulk of the activity was detected in the supernatant. Highest activities were observed after treatment with Triton X-100. The bulk of the AcP activity remained bound to the pellet, if suspension and fractionation of the homogenized stratum corneum were performed in acetate buffer in the range between pH 4.0-5.0, probably due to ionic or hydrophobic interactions. AcP activity was totally lost if homogenates or fractions were stored frozen at -20 degrees C in buffers with pH values lower than 4.0. Triton X-100 extracts from whole skin, epidermis, stratum corneum, cultured skin fibroblasts and leukocytes were compared by isoelectric focusing. Extracts from whole skin, epidermis and stratum corneum yielded almost identical patterns with one main AcP activity band at pI of 5.65, whereas a second pronounced band from whole skin behaved similarly to one band from cultured skin fibroblasts and leukocytes (pI 6.1). The prominent band from extracts of stratum corneum and epidermis was not observed in extracts of skin fibroblasts and leukocytes. Hence, we conclude that stratum corneum and epidermis contain a tissue-specific AcP.     

Characteristics of the epidermis and stratum corneum of hairless mice with experimentally induced diabetes mellitus

Diabetes mellitus induces many pathophysiologic changes in the skin. Even so, dermatologists still lack an animal model of diabetes that enables the direct evaluation of the various functional properties of the skin. Our group induced two types of an experimental type 1 diabetes model in hairless mice by administering either streptozotocin or alloxan, in order to examine the properties of the stratum corneum and epidermis of these animals. The plasma glucose concentrations of the mice at 3 wk after their i.v. injection were significantly higher than those of control mice (streptozotocin, 3.2-fold; alloxan, 3.7-fold). The stratum corneum water content was significantly reduced in both types of diabetic mice, whereas the transepidermal water loss remained unchanged. The amino acid content with normal epidermal profilaggrin processing was either normal or elevated in the stratum corneum of the streptozotocin-treated mice. In contrast, the stratum corneum triglyceride content in the streptozotocin-treated mice was significantly lower than the control level, even though the levels of ceramides, cholesterols, and fatty acids in the stratum corneum were all higher than the control levels. The streptozotocin-treated group also exhibited decreases in basal cell proliferation and epidermal DNA content linked with an increase in the number of corneocyte layers in the stratum corneum, suggesting that the rates of epidermal and stratum corneum turnover were slower in the streptozotocin-treated animals than in the normal controls. In contrast, there were no remarkable changes in any of the epidermal differentiation marker proteins examined. This finding in diabetic mice, namely, reduction in both the epidermal proliferation and stratum corneum water content without any accompanying impairment in the stratum corneum barrier function, is similar to that found in aged human skin. Our new animal model of diabetes will be useful for the study of diabetic dermopathy as well as the mechanisms of stratum corneum moisturization.     

Loss of water from the stratum corneum induces epidermal DNA synthesis in hairless mice

Abstract Many clinical studies have shown that low humidity has a deleterious effect on skin, but the mechanisms involved are poorly understood. To clarify the changes that occur in skin, we examined epidermal cell proliferation in mice kept in a dry (relative humidity < 10%) or a moist (relative humidity > 90%) environment. In animals exposed to low humidity, epidermal DNA synthesis started to increase within 12 h, reaching twice the original level, and the increased level was maintained for up to 5 days. The transepidermal water loss (TEWL) of mice kept for 12 h in the dry environment was the same as that of mice kept in the moist enviroment, but the skin conductance was lower. The increase in epidermal DNA synthesis following exposure to the dry environment was inhibited by topical application of petrolatum. It is concluded that loss of water from the stratum corneum induces epidermal cell proliferation within 12 h, and this change occurs in the absence of apparent cutaneous barrier dysfunction. Received: 20 April 1998 / Received after revision: 2 July 1998 / Accepted: 31 July 1998     

Physical and physiological effects of stratum corneum tape stripping

Background/aims: Tape stripping of human stratum corneum has been performed to measure stratum corneum mass, barrier function, drug reservoir and percutaneous penetration. However, the technique itself requires further development to facilitate interpretation.
Methods: In this study we quantified stratum corneum (SC) tape stripping and water kinetic parameters utilizing three types of adhesive tapes, in an in vivo randomized clinical trial. Stratum corneum was tape stripped, and the mass of SC removed by each tape was quantified utilizing a protein assay. Transepidermal water loss (TEWL) was measured and barrier disruption and SC water kinetics calculated. Three commonly utilized acrylate adhesive tapes were utilized and a comparison made between them.
Results: Each type of tape successfully stripped the stratum corneum, but the rayon tape did not induce SC barrier disruption. Neither the type of tape nor the site stripped significantly influenced the mass of SC removed. Water kinetic parameters did not differ significantly for the tapes that did induce barrier disruption. Individual variation in barrier disruption to water following tape stripping was demonstrated.
Conclusion: The tapes utilized removed a similar amount of SC. The tapes have a different propensity to cause barrier disruption. Some individuals do not demonstrate increased TEWL despite an equivalent mass of SC being removed compared to those who do show a response.     

Effects of glycolic acid on desquamation-regulating proteinases in human stratum corneum

In order to investigate the mechanism of glycolic acid (GA) function in human stratum corneum, we monitored changes in cathepsin D-like (CD) and chymotrypsin-like (SCCE) proteinases for 3 weeks following topical GA application (50% w/v, pH 0.9) for 30 min to human skin. In the early phase, weakened stratum corneum cohesion in the lower layers was observed on day 2 and the amount of active CD in the upper layer of the stratum corneum was significantly decreased from 30 min until day 2, whereas that in the lower layer remained normal. In contrast, the amount of active SCCE showed no change during the experimental period. The surface pH of the stratum corneum drastically decreased to pH 2 at 30 min and slightly recovered to around pH 3 until 1 day after treatment. From 9 to 19 days, a decrease in corneocyte cell area and a remarkable long-term increase in the amount of active CD in the upper layer were observed. In an in vitro study, the activities of desquamation-regulating proteinases were shown to have remarkably increased at around pH 3, due to activation of CD at its optimal pH. These results suggest that GA functions via at least two different mechanisms, acute activation of CD in the lower layer by acidification around pH 3, along with inactivation of CD in the upper layer, and long-term enhancement of de novo CD production in the few weeks following GA treatment.     

Mode of action of glycolic acid on human stratum corneum: ultrastructural and functional evaluation of the epidermal barrier

Alpha-hydroxy acids (AHA) such as glycolic acid have recently been used extensively in cosmetic and dermatological formulas. In low concentration (2– 5%) glycolic acid is believed to facilitate progressive weakening of cohesion of the intercellular material of the stratum corneum (SC), resulting in uniform exfoliation of its outermost layers (the stratum disjunctum). Since thinning of the SC as well as changes of intercellular lipids could theoretically compromise the barrier functions of the skin, we investigated the mode of AHA action on the SC to determine whether enhanced desquamation compromises the barrier structures of the SC and changes transepidermal water loss (TEWL) values. Electron microscopy of the epidermis biopsied from the volar forearm of human volunteers after 3 weeks of treatment with a 4% glycolic acid formulation twice daily was employed to evaluate 1) epidermal morphology and thickness of the SC, (2) the lamellar body and SC lipid bilayer organization, and (3) desquamative events based on degradation of desmosomes. TEWL values and SC hydration were recorded prior to and at the end of the study. Electron microscopy revealed no ultrastructural changes in the nucleated layers of the epidermis. The lamellar body (LB) secretory system in the stratum granulosum (SG), and intercellular lipid lamellae in the SC in both vehicle- and glycolic acid-treated samples were comparable to normal human SC. Within the SC, enhanced desmosomal breakdown, promoting loss of cohesion and desquamation, was restricted to the stratum disjunctum while desmosomes of the stratum compactum were unaffected. Treated areas displayed histologically, a more compact appearing SC. TEWL values remained unchanged in glycolic acid- and vehicle-treated skin. Our findings indicate that the barrier structures of the SC are not disrupted by glycolic acid formulations at the concentration used. One of the mechanism of action of AHA on the SC seemed to be a „targeted“ desmosomal (corneosomal) action without compromising the barrier structures of the skin. Received: 20 November 1996     

Structural investigations of human stratum corneum by small-angle X-ray scattering.

The structure of human stratum corneum was investigated with small-angle X-ray scattering (SAXS). At room temperature the scattering curve was characterized by a strong intensity at low scattering vector (Q less than 0.8 nm-1) and two complicated diffraction peaks originating from a lamellar structure of the lipids. The lamellar lipid structure in the stratum corneum transformed to a disordered structure between 65 degrees C and 75 degrees C, the same temperature region at which a thermal lipid transition occurred. After cooling down to room temperature a recrystallization of at least a part of the lipids took place, after which only one unit cell with a repeat distance of 13.4 nm could be detected. Comparison of the scattering curve of the stratum corneum after crystallization with the scattering curve of the stratum corneum before recrystallization leads to the conclusion that in the original curve the lipids are arranged in two unit cells with repeat distances of 6.4 nm and 13.4 nm. From model calculations it appears that the latter unit cell consists of more than one bilayer. The scattering curves of stratum corneum hydrated to various levels were measured. A change in the water content of stratum corneum between 6% w/w and 60% w/w (fully hydrated) did not result in swelling of the bilayers, but the scattering curve obtained with stratum corneum hydrated to 60% w/w differed from those at lower hydration levels: the scattering curve at 60% w/w showed only the diffraction peaks corresponding to a unit cell with a repeat distance of 6.4 nm. This observation implies that the ordering of a part of the lipids is reduced at very high water contents, which may explain the strong penetration-enhancing effects of water in the stratum corneum.     

Ethnicity and stratum corneum ceramides

Background The barrier function of the skin is dependent on an optimal composition of the stratum corneum lipids, exemplified by the altered lipid profile in patients with atopic eczema (AE). Differences in the global prevalence of AE point to the environment as an important factor in AE. Studies on filaggrin point to a genetic aspect in AE. The influence of environment and genes needs to be explored. Objectives To investigate possible differences in stratum corneum lipids between different healthy ethnicities living in the same environment. Methods Healthy participants without any major skin diseases were enrolled in the study. Twenty‐five participants of Asian origin (Asians), 18 of African origin (Africans) and 28 of Danish origin (white‐skinned), all students at universities in the Copenhagen area of Denmark, had the ceramide profile of their stratum corneum examined using the cyanoacrylate method and analysed using high‐performance thin layer chromatography. Results For the ceramide/cholesterol ratio we found statistically significant differences between groups, with Asians having the highest ratio (P < 0·001 as compared with both white‐skinned individuals and Africans), white‐skinned individuals having intermediate values, and Africans having the lowest values. No statistically significant differences were found between any of the ceramide subgroups. Conclusions We found different ceramide/cholesterol ratios in comparable groups of different ethnicity, pointing to unknown genetic differences.     

The role of proteases in stratum corneum: involvement in stratum corneum desquamation

The effects of protease inhibitors on cell dissociation were studied in vitro in order to examine the involvement of proteases in stratum corneum desquamation. Stratum corneum sheet (peeled from human backs after sunburn) was incubated in a detergent mixture containing 8 mM N,N-dimethyldodecylamine oxide, 2 mM sodium lauryl sulphate and 60 g/ml kanamycin with or without protease inhibitors, and the number of released cells was counted after incubation for 48 h. Cell dissociation was inhibited strongly by antipain or aprotinin, but not at all by N-[N-(l-3-transcarboxyoxiran-2-carbonyl)-l-leucyl]-agmatin, N-ethylmaleimide or pepstatin, which suggests that only serine proteases are associated with desquamation. Furthermore, leupeptin and chymostatin each reduced cell dissociation about half as effectively as aprotinin or antipain, while a mixture of leupeptin and chymostatin prevented stratum corneum dissociation as potently as antipain or aprotinin. In addition, the activity of chymotrypsin-like protease in scaly skin was higher than that in normal skin, as we have previously found for trypsin-like protease. These results suggest that both trypsin-like and chymotrypsin-like serine proteases are involved in stratum corneum desquamation.     

Detection of the action of salicylic acid on the normal stratum corneum

The undoubted clinical usefulness of salicylic acid contrasts markedly with its barely detectable action on normal skin. In this study we have attempted to extend our understanding of the action of salicylic acid by monitoring its effect on the stratum corneum using newly developed techniques for assessing desquamation, as well as using conventional methods for measuring other aspects of epidermal activity. Our findings have succeeded in demonstrating that salicylic acid enhances the shedding of corneocytes and we suggest that this compound in some way decreases corneocyte to corneocyte cohesion. We have confirmed that salicylic acid has no effect on the mitotic activity of the normal epidermis.     

Emollient molecule effects on the drying stresses in human stratum corneum

Background  Emollient molecules are widely used in skin care formulations to improve skin sensory properties and to alleviate dry skin but little is understood regarding their effects on skin biomechanical properties. Objectives  To investigate the effects of emollient molecules on drying stresses in human stratum corneum (SC) and how these stresses are related to SC components and moisture content. Methods  The substrate curvature method was used to measure the drying stresses in isolated SC following exposure to selected emollient molecules. While SC stresses measured using this method have the same biaxial in vivo stress state and moisture exchange with the environment, a limitation of the method is that moisture cannot be replenished by the underlying skin layers. This provides an opportunity to study the direct effects of emollient treatments on the moisture content and the components of the SC. Attenuated total reflectance Fourier transform infrared spectroscopy was used to determine the effects of emollient molecules on SC lipid extraction and conformation. Results  Emollient molecules resulted in a complex SC drying stress profile where stresses increased rapidly to peak values and then gradually decreased to significantly lower values compared with the control. The partially occlusive treatments also penetrated into the SC where they caused extraction and changes in lipid conformation. These effects together with their effects on SC moisture content are used to rationalize the drying stress profiles. Conclusions  Emollient molecules have dramatic effects on SC drying stresses that are related to their effects on intercellular lipids and SC moisture content.     

The changes of stratum corneum interstices and calcium distribution of follicular epithelium of experimentally induced comedones (EIC) by oleic acid*

Abstract Abnormal follicular keralinization is important for comedo formation in acne, but the precise mechanism is not known. A recent report about acne vulgaris suggested that an impaired water barrier function may be responsible for comedo formation, since the barrier dysfunction is accompanied by hyperkeratosis of the follicular epithelium. Furthermore, the integrity of the water barrier is crucial for the maintenance of the epidermal calcium gradient. Yet stratum corneum intercellular lipid structures, a major factor of the skin barrier function, and calcium distribution in the follicular epithelium of comedones, were not reported. To see SC intercellular lipid and calcium distribution of the follicular epithelium of comedo by electron microscopy, we applied oleic acid on the inner surface of the ear of New Zealand white rabbits to induce comedones, and then we obtained specimens and performed osmium and ruthenium tetroxide postfixation and calcium ion-capture cytochemical procedure. We found incomplete lipid bilayer structures, prominent dilatation of lacunar domains and the loss of follicular epidermal calcium gradient in experimentally induced comedones. From our results, we suggest that the permeability barrier disruption in oleic-acid-applied follicular epithelium can be induced by the changes of SC intercellular membrane structures and lacunar dilatation, and the calcium gradient is lost, so follicular epithelial proliferation and hyperkeratosis can be induced and then comedo formation occurs.