Murine Stratum Corneum Research Articles

Decreased Levels of Covalently Bound Ceramide Are Associated with Ultraviolet B-Induced Perturbation of the Skin Barrier

Although ultraviolet B (UVB) irradiation perturbs the skin barrier, little is known about the mechanism(s) with respect to the metabolism of ceramide (Cer). We examined changes in intercellular lipids in murine stratum corneum following UVB irradiation. A single UVB (75 mJ per cm(2)) irradiation caused a significant increase in transepidermal water loss, which plateaued at day 4. In parallel, covalently bound Cer was significantly decreased with the greatest decrease at days 3-4. In contrast, the levels of other free, non-bound lipids (including Cer or acylceramides) were significantly increased for Cer, or remained unchanged at day 4 compared with non-irradiated controls. RT-PCR analysis demonstrated a significant decrease in mRNA encoding transglutaminase-1 (TGase1). The peak occurred 2-4 d after a single UVB irradiation, a time when covalently bound Cer was significantly downregulated in concert with the disruption of the skin barrier. Furthermore, UVB-induced epidermal hyperplasia occurred to the greatest extent between 2 and 4 d following UVB irradiation. These results suggest that decreases in covalently bound Cer in the stratum corneum are mediated via the downregulation of TGase-1 as well as by the rapid induction of epidermal hyperplasia, which is attributable to the perturbation of the skin barrier induced by UVB irradiation.

Generation of Free Fatty Acids from Phospholipids Regulates Stratum Corneum Acidification and Integrity

There is evidence that the "acid mantle" of the stratum corneum is important for both permeability barrier formation and cutaneous antimicrobial defense. The origin of the acidic pH of the stratum corneum remains conjectural, however. Both passive (e.g., eccrine/sebaceous secretions, proteolytic) and active (e.g., proton pumps) mechanisms have been proposed. We assessed here whether the free fatty acid pool, which is derived from phospholipase-mediated hydrolysis of phospholipids during cornification, contributes to stratum corneum acidification and function. Topical applications of two chemically unrelated secretory phospholipase sPLA2 inhibitors, bromphenacylbromide and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, for 3 d produced an increase in the pH of murine skin surface that was paralleled not only by a permeability barrier abnormality but also altered stratum corneum integrity (number of strippings required to break the barrier) and decreased stratum corneum cohesion (protein weight removed per stripping). Not only stratum corneum pH but also all of the functional abnormalities normalized when either palmitic, stearic, or linoleic acids were coapplied with the inhibitors. Moreover, exposure of intact murine stratum corneum to a neutral pH for as little as 3 h produced comparable abnormalities in stratum corneum integrity and cohesion, and further amplified the inhibitor-induced functional alterations. Furthermore, short-term applications of an acidic pH buffer to inhibitor-treated skin also reversed the abnormalities in stratum corneum integrity and cohesion, despite the ongoing decrease in free fatty acid levels. Finally, the secretory-phospholipase-inhibitor-induced alterations in integrity/cohesion were in accordance with premature dissolution of desmosomes, demonstrated both by electron microscopy and by reduced desmoglein 1 levels in the stratum corneum (shown by immunofluorescence staining and visualized by confocal microscopy). Together, these results demonstrate: (i) the importance of phospholipid-to-free-fatty-acid processing for normal stratum corneum acidification; and (ii) the potentially important role of this pathway not only for barrier homeostasis but also for the dual functions of stratum corneum integrity and cohesion.

Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O 3) alone and in combination. Whereas a significant depletion of α-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O 3 for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O 3×2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O 3 at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.

48] High-pressure liquid chromatography analysis of ozone-induced depletion of hydrophilic and lipophilic antioxidants in murine skin

Publisher Summary Ozone (O 3 ) is best known for its role in the stratosphere where it filters out much of the short-wave spectrum of solar ultraviolet (UV) radiation that would otherwise penetrate the earth. However, it is also well-known that this protective “ozone layer,” if placed at ground level (troposphere), would be highly toxic and would react with various molecules susceptible to oxidative attack. Because of the breakdown of the stratospheric ozone layer in conjunction with industrial emission, tropospheric ozone has become a major environmental pollutant. Increased exposure to this powerful oxidant has various health implications currently under investigation. Ozone is well known to exert profound effects on the respiratory tract. Studies suggest that ozone also induces biochemical changes in murine skin. Due to its barrier structure and function, the skin is highly susceptible to external oxidative agents. More specifically, the outermost layer of the skin, the stratum corneum (SC), has been identified as the primary target for ozone-induced damage. This chapter discusses a highly sensitive method for detecting antioxidants in murine SC before and after in vivo ozone exposure.

Vitamin C, Uric Acid, and Glutathione Gradients in Murine Stratum Corneum and their Susceptibility to Ozone Exposure

The stratum corneum has been recognized as the main cutaneous oxidation target of atmospheric ozone (O3), a major part of photochemical smog. This study reports the presence and distribution of vitamin C, glutathione, and uric acid in murine stratum corneum, and evaluates their susceptibility to acute environmental exposure to O3. Based on tape stripping and a modified extraction method with high performance liquid chromatography electrochemical analysis, we detected vitamin C (208.0 +/- 82.5 pmol per 10 consecutive pooled tapes), glutathione (283.7 +/-96.3), and uric acid (286.4 +/-47.1) in murine stratum corneum as compared with only 16.5 +/- 1.4 pmol alpha-tocopherol. Vitamin C, glutathione (both p < 00.001), and urate (p < 0.01) were found to exhibit a gradient with the lowest concentrations in the outer layers and a steep increase in the deeper layers. To investigate the effect of O3 exposure on hydrophilic antioxidants, we exposed SKH-1 hairless mice to O3 concentrations of 0, 0.8, 1, and 10 p.p.m., and stratum corneum was analyzed before and after exposure. Whereas mock exposure with 0 p.p. m. for 2 h had no significant effect, O3 doses of 1 p.p.m. for 2 h and above showed depletion of all three antioxidants. Vitamin C was decreased to 80% +/- 15% of its pretreatment content (p < 0.05), GSH to 41% +/- 24% (p < 0.01), and uric acid to 44% +/- 28% (p < 0.01). This report demonstrates the previously unrecognized role of hydrophilic antioxidants in the stratum corneum and provides further evidence that O3 induces oxidative stress in this outer skin layer.

Depletion of Human Stratum Corneum Vitamin E: An Early and Sensitive In Vivo Marker of UV Induced Photo-Oxidation

As the outermost barrier of the body, the stratum corneum (SC) is frequently and directly exposed to a pro-oxidative environment, including ultraviolet solar radiation (UVR). Therefore, we hypothesized that the SC is susceptible to UVR induced depletion of vitamin E, the major lipophilic antioxidant. To test this, we investigated (i) the susceptibility of SC tocopherols to solar simulated UVR in hairless mice, (ii) the baseline levels and distribution patterns of tocopherols in human SC, and (iii) the impact of a suberythemogenic dose of solar simulated UVR on human SC tocopherols. SC tocopherol levels were measured by high performance liquid chromotography analysis of SC extracts from tape strippings. In murine SC, overall tocopherol concentrations were determined, whereas in human SC, 10 consecutive layers were analyzed for each individual. The results on SC tocopherols demonstrated (i) their concentration dependent depletion by solar simulated UVR in hairless mice; (ii) a gradient distribution within untreated human SC, with the lowest levels at the surface (alpha-tocopherol 6.5 +/- 1.4 pmol per mg, and gamma-tocopherol 2.2 +/- 1.3 pmol per mg) and the highest levels in the deepest layers (alpha-tocopherol 76 +/- 12 pmol per mg, and gamma-tocopherol 7.9 +/- 3.7 pmol per mg, n = 10; p < 0.0001); and (iii) the depletion of tocopherols in human SC by a single suberythemogenic dose of solar simulated UVR (alpha-tocopherol by 45%, and gamma-tocopherol by 35% as compared with controls; n = 6; both p < 0.01). These results demonstrate that the SC is a remarkably susceptible site for UVR induced depletion of vitamin E.

Localization of glucocerebrosidase within murine stratum corneum

Localization of glucocerebrosidase within murine stratum corneum

Membrane Structures in Normal and Essential Fatty Acid-Deficient Stratum Corneum: Characterization by Ruthenium Tetroxide Staining and X-Ray Diffraction

Despite the importance of intercellular lamellar bilayers for stratum corneum (SC) barrier function, knowledge about the structure of these bilayers is limited due to their poor visualization and/or retention. Whereas substitution of ruthenium tetroxide (RuO4) for osmium tetroxide fixation provides clear images of these bilayers, the usefulness of RuO4 has been limited by its slow penetration and cytotoxicity. Utilizing a new fixation protocol for RuO4, we obtained clear images of lamellar domains at all levels of murine SC. Computer-aided image reconstructions demonstrated a lamellar spacing of 129 +/- 2 A, which agreed with x-ray diffraction data from parallel, unfixed samples (131 +/- 2 A), a spacing not affected by hydration. Furthermore, novel structures were seen in the intercellular spaces of normal SC. Finally, in murine essential fatty acid deficiency (EFAD), the overall lamellar spacing is comparable to normal [127 +/- 7 A by computer transform vs. 131.9 +/- 2 A (hydrated) and 129.6 +/- 2.2 A (dry) by x-ray diffraction]. Yet, these domains are structurally abnormal, displaying regions with either an excess or absence of lamellae. The new RuO4 protocol provides quantitative information about SC lamellar dimensions and morphologic abnormalities in bilayer distribution and substructure in EFAD stratum corneum that are not detected by either x-ray diffraction or computer-aided image reconstruction. Thus, the barrier abnormality in EFAD stratum corneum can be ascribed either to focal depletion of lamellae or abnormalities in lamellar substructure.

Localization of Lipid Microdomains and Thermal Phenomena in Murine Stratum Corneum and Isolated Membrane Complexes: An Electron Spin Resonance Study

The intercellular region of the stratum corneum can be isolated in the form of membrane complexes (intercellular lipids and adjacent cornified envelopes) which are devoid of the cytoplasmic components. In this study the temperature induced phase transitions and corresponding lipid domain reorganization in isolated stratum corneum (SC) sheets and SC membrane complexes (SCM) were determined using the electron spin resonance (ESR) spin probe technique. The spin probe perdeuterated di-tert-butyl nitroxide (pdDTBN) in SC and SCM revealed less well-defined physiologic phase transitions in the SCM and a more polar lipid domain in SC. However, the ESR results show the coexistence of highly ordered domains (immobilized spin probe) and less viscous domains in intact SC, which persist in SCM. Above approximately 20 degrees C the spin probe is dissolved in a highly disordered (isotropic) lipid domain in both SC and SCM. In both solvent extracted SC and SCM, the spin probe is dissolved in a highly ordered lipophilic domain associated with the lipids complexed to the corneocyte envelope and exhibiting no thermal transitions between -23 degrees to 60 degrees C. Further, the amount of mobile spin probe is related to the amount of residual lipid. An unexpected finding was the apparent reduction of the spin probe in solvent extracted SCM, suggesting the presence of a previously unrecognized free radical reducing mechanism in these sites. The mobilities of the spin probe when dissolved in model lipids, non-hydroxy, and hydroxy containing ceramides and cholesteryl oleate, differed significantly from those observed in SC or SCM. These studies demonstrate the usefulness of ESR for the localization and characterization of lipid microenvironments in the stratum corneum.

Mechanism of percutaneous penetration enhancement: effect of n-alkanols on the permeability barrier of hairless mouse skin

To examine the action of alcohols on skin barrier function, we have: (a) determined, in vitro, how pretreatment of hairless mouse skin with a homologous series of n-alkanols (C 2-C 12) enhances the transport of a model penetrant (nicotinamide); (b) measured the skin uptake and permeation of the alkanols themselves during the pretreatment period; and (c) correlated the data from these two sets of experiments with biophysical evaluation of the pretreated murine stratum corneum using Fourier transform infrared spectroscopy (FT-IR). Following 3 or 6 h pretreatment, nicotinamide flux was increasingly enhanced with increasing alkanol carbon number up to C 6. The effect of octanol was similar to that of hexanol, but decanol was less efficacious and dodecanol continued this diminishing trend. While skin uptake of the alkanols increased linearly with increasing lipophilicity (i.e., increasing chain length), the cumulative penetration (in 6 h) of the homologs was a parabolic function of C n similar to the nicotinamide enhancement profile. FT-IR analysis of ethanol-pretreated hairless mouse skin revealed considerable extraction of stratum corneum intercellular lipids; the appearance of these lipids in the pretreatment liquid could also be measured spectroscopically. The same extraction phenomenon was also apparent when pretreatment was performed with butanol and octanol. For these homologues, removal of stratum corneum lipids was shown unequivocally by FT-IR through the use of perdeuterated alkanols, thereby separating the C-H(D) signals from intercellular lipid and penetration enhancer. Lipid extraction, therefore, is implicated as the mechanism by which alkanols promote transdermal penetration. Because of the efficiency of lipid removal in these experiments, it was not possible to determine if additional, and possibly more subtle, effects (e.g., “fluidization” of stratum corneum lipid domains) are also induced by alkanols.

Membrane Structural Alterations in Murine Stratum Corneum: Relationship to the Localization of Polar Lipids and Phospholipases

Membrane Structural Alterations in Murine Stratum Corneum: Relationship to the Localization of Polar Lipids and Phospholipases

Membrane Structural Alterations in Murine Stratum Corneum: Relationship to the Localization of Polar Lipids and Phospholipases

During the formation of the mammalian epidermal permeability barrier, lipids are sequestered in the stratum corneum intercellular spaces, transforming from a relatively polar lipid mixture to predominantly nonpolar species. Certain lipid catabolic enzymes, which co-localize with these lipids, may regulate this process. In order to localize the sites within the outer epidermis where polar lipids are catabolized, and their relationship to the alterations in membrane structure that occur in these layers, we compared the biochemical localization of polar lipids, the ultrastructure, and freeze-fracture morphology, as well as the localization of phospholipases within the outer epidermis. Both histochemical staining of frozen sections and biochemical studies of protease- and tape-stripped whole stratum corneum demonstrated small amounts of polar lipids in the stratum compactum, while in contrast, the stratum disjunctum was devoid of both phospholipids and glycosphingolipids. Phospholipase activity was present within lamellar bodies, among secreted lamellar body disks at the granular-cornified layer interface, and within the intercellular spaces of the stratum compactum. Both the depletion of polar lipids from the stratum compactum and deletion of these substances from the stratum disjunctum correlated with sequential changes in membrane structure observed by transmission electron microscopy and freeze-fracture. Thus, a phospholipase-mediated attack on phospholipids (with a parallel assault by other lipid catabolic enzymes on other polar species), may induce both the initial fusion and elongation of lamellar body disks and the subsequent formation of the hydrophobic membrane bilayers found in the mid-to-outer stratum corneum. These studies also may require modification of traditional views of the stratum corneum as a metabolically inert tissue, revealing its intercellular lipid domains to be partially in an active state of flux.

Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffraction study.

The lipid of the outermost layer of the skin is confined largely to the extracellular spaces surrounding the corneocytes of the stratum corneum where it forms a multilamellar adhesive matrix to act as the major permeability barrier of the skin. Knowledge of the molecular architecture of these intercellular domains is important for understanding various skin pathologies and their treatment, percutaneous drug delivery, and the cosmetic maintenance of the skin. We have surveyed by X-ray diffraction the structure of the intercellular domains and the extracted lipids of murine stratum corneum (SC) at 25, 45, and 70 degrees C which are temperatures in the vicinity of known thermal phase transitions [Rehfeld, S. J., & Elias, P. M. (1982) J. Invest. Dermatol. 79, 1-3]. The intercellular domains produce lamellar diffraction patterns with a Bragg spacing of 131 +/- 2 A. Lipid extracted from the SC and dispersed in excess water does not produce a simple lamellar diffraction pattern at any temperature studied, however. This and other facts suggest that another component, probably a protein, must be present to control the architecture of the intercellular lipid domains. We have also obtained diffraction patterns attributable to the protein envelopes of the corneocytes. The patterns suggest a beta-pleated sheet organizational scheme. No diffraction patterns were observed that could be attributed to keratin.