Cannabinoid receptors 1 and 2 oppositely regulate epidermal permeability barrier status and differentiation

Cannabinoid receptors (CBR) 1 and 2 have been implicated in keratinocyte differentiation/proliferation. How CB receptors affect epidermal permeability barrier and stratum corneum structure and function remains unclear. Permeability barrier abrogation was induced by sequential tape-stripping of the SC and assessed in both CB1R and CB2R knockout (-/-) mice in comparison with wild-type (+/+) littermates. Absence of CB1R delays permeability barrier recovery, while the latter was found to be accelerated in CB2R -/- mice. While increased lamellar body (LB) secretion is observed in CB2R -/- mice accounting for the enhanced recovery, CB1R -/- animals display strong alterations in lipid bilayer structures. Markers for epidermal differentiation (i.e. filaggrin, loricrin and involucrin) and terminal differentiation (i.e. TUNEL assay and caspase-14 activation) were respectively decreased and increased in CB1R and CB2R -/- mice. Surprisingly, CB1R agonist treatment of human cultured keratinocytes increases mRNA of p21 and cytokeratin 1 and 10 and decreases cyclin D1 but protein levels remained unchanged. Such paradox could partially be explained by the increase in non-phosphorylated-4E-BP1, an inhibitor of mRNA translation, following CB1R agonist treatment. Altogether, these observations put forward the importance and the complexity of cannabinoid signalling for the regulation of permeability barrier and epidermal differentiation.     

Integrity of the permeability barrier regulates epidermal Langerhans cell density

Summary Previous studies have shown that barrier requirements regulate epidermal liquid and DNA synthesis. In the present study, we examined the possibility that the integrity of the permeability barrier influences epidermal Langerhans cells involved with the immune response. Barrier disruption was achieved by treatment of human skin with acetone, sodium dodecylsulphate (SDS), or tape stripping, until a 10–20-fold increase in transepidermal water loss was achieved. Serial biopsies were performed 6–168 after treatment, and Langerhans cells were complexed with anti-CD1a (Leu6) or S-l00 antibodies, and visualized with an immunoperoxidase technique. Acetone treatment resulted in an increase in epidermal Langerhans cell density, reaching a maximum of 94% over control (P < 0.01) by 24 and 48 h post-treatment. Following SDS treatment or tape stripping, epidermal Langerhans cell density was increased by 100 and 175% (P < 0.01), respectively. There was a linear correlation between the degree of barrier disruption and the increase in epidermal Langerhans cell density. Studies with the Ki-S3 proliferation-associated nuclear antigen revealed a two- to threefold increase in epidermal proliferation after barrier disruption. The time curves of the increase in Langerhans cell density and the increase in epidermal proliferation were similar, suggesting that there was a coordinate regulation. In contrast with our previous studies employing patch test reactions to allergens or irritants, disruption of barrier function neither resulted in an increased dermal Langerhans cell density, nor influenced T lymphocytes (CD3⁺. Leu4⁺). Macrophages (KiM8⁺), ICAM-1 or ELAM-1 expression in the skin. In addition, barrier disruption did not result in either dermal inflammation or epidermal spongiosis. In summary these findings support our hypothesis that the permeability barrier influences epidermal Langerhans cell density, which is involved in maintaining an immunological barrier.     

Cutaneous barrier repair and pathophysiology following barrier disruption in IL-1 and TNF type I receptor deficient mice

Disruption of the permeability barrier elicits a homeostatic repair response which rapidly restores barrier function while repeated barrier perturbation results in cutaneous pathology. In response to barrier disruption there is a marked increase in epidermal TNF-alpha and IL-1 production. To determine the potential role of TNF and IL-1 in mediating the cutaneous changes that occur following barrier disruption we compared the kinetics of barrier recovery and the degree of epidermal hyperplasia and cutaneous inflammation in TNF type I (p55) receptor and IL-1 receptor type I (p80) deficient mice. No abnormalities in epidermal morphology were observed with light or electron microscopy in receptor deficient mice. Under baseline conditions epidermal barrier function was unchanged in receptor deficient mice. Following barrier disruption the kinetics of barrier recovery were similar in control vs TNF receptor deficient mice regardless if the barrier was disrupted by acetone treatment, SDS treatment, or tape stripping. In contrast, barrier recovery was slightly but significantly accelerated regardless of the method of barrier disruption in IL-1 receptor deficient mice. The degree of epidermal hyperplasia and cutaneous inflammation following repeated barrier disruption was similar in control, TNF receptor, and IL-1 receptor deficient mice. The present study demonstrates that barrier recovery is not delayed and the degree of epidermal hyperplasia and inflammation are not altered in either TNF receptor or IL-1 receptor deficient mice, indicating that neither TNF nor IL-1 alone are essential for either barrier repair or the cutaneous pathology induced by barrier perturbation. Whereas the increase in IL-1 following barrier disruption may delay components of the repair response, whether either TNF-alpha or IL-1 regulate aspects of the homeostatic response remains unresolved.     

Role of the epidermal barrier in atopic dermatitis

The skin's permeability barrier protects against extensive water loss and prevents the entry into the skin of harmful substances like irritants, allergens and microorganisms. The permeability barrier is mainly located in the stratum corneum and consists of corneocytes and a lipid‐enriched intercellular domain. The barrier is formed during epidermal differentiation. In atopic dermatitis the skin barrier is disturbed already in non‐lesional skin. The disturbed skin barrier allows the entry of environmental allergens from house dust mites, animal dander and grass pollen into the skin. In predisposed individuals these allergens may trigger via immunologic pathways the inflammation of atopy. The causes for the disturbed epidermal skin barrier are changes in skin lipids and in epidermal differentiation, in particular filaggrin mutations. Filaggrin mutations lead to a disturbed skin barrier and dry skin which are hallmarks in atopic dermatitis. Therapeutic agents influence the skin barrier differently; topical therapy with potent corticosteroids does not lead to the repair of the barrier in atopic dermatitis, whereas therapy with the calcineurin inhibitors and lipid‐containing emulsions support barrier repair.     

Skin barrier function, epidermal proliferation and differentiation in eczema

Skin permeability barrier function is impaired in eczema, particularly in contact and atopic dermatitis (AD). In contact dermatitis disruption of the barrier by irritants and allergens is the primary event, followed by sensitization, inflammation, increased epidermal proliferation and changes in differentiation. Genetically impaired skin barrier function is already present in non-lesional and more pronounced in lesional skin in AD. Increased epidermal proliferation and disturbed differentiation, including changes in lipid composition, cause impaired barrier function in AD. Defective permeability barrier function enables the enhanced penetration of environmental allergens into the skin and initiates immunological reactions and inflammation. Barrier dysfunction is therefore crucially involved in the pathogenesis of AD. The atopic syndrome represents a genetically impaired skin barrier function as well as impaired nasal, bronchial, and intestinal mucous membranes leading to AD, allergic rhinitis, bronchial asthma or aggravation of AD. Common treatment strategies for eczema include the application of lipid-based creams and ointments, which aim toward the restoration of the defective permeability barrier, thus helping to normalize proliferation and differentiation.     

pH in nature,humans and skin

The pH plays an important physiological role in nature and humans. pH varies from 1 to 8 in human organs with tight regulation in blood and epithelia of barrier organs. The physiological pH of the stratum corneum is 4.1–5.8 and several mechanisms contribute to its formation: filaggrin degradation, fatty acid content, sodium‐hydrogen exchanger (NHE1) activation and melanosome release. First, the acidic pH of the stratum corneum was considered to present an antimicrobial barrier preventing colonization (e.g. by Staphylococcus aureus and Malassezia). Later on, it was found that the pH influences skin barrier function, lipid synthesis and aggregation, epidermal differentiation and desquamation. Enzymes of ceramide metabolism (e.g. β‐glucocerebrosidase or acid sphingomyelinase) as well as proteases (e.g. chymotryptic enzyme or cathepsin D linked to epidermal differentiation and desquamation) are regulated by the pH. Experimental disruption of the physical barrier leads to an increase of pH, returning to normal levels only after many hours. Inflammatory skin diseases and diseases with an involvement of the epidermis exhibit a disturbed skin barrier and an increased pH. This is known for atopic dermatitis, irritant contact dermatitis, ichthyosis, rosacea and acne, but also for aged and dry skin. Normalizing the pH by acidification through topical treatment helps to establish a physiological microbiota, to repair skin barrier, to induce epidermal differentiation and to reduce inflammation.     

Topical treatment with thiazolidinediones, activators of peroxisome proliferator-activated receptor-gamma, normalizes epidermal homeostasis in a murine hyperproliferative disease model

In a murine model of epidermal hyperplasia reproducing some of the abnormalities of several common skin disorders, we previously demonstrated the antiproliferative and pro-differentiating effects of peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and liver X receptor activators. Unlike other subgroups of PPAR activators, thiazolidinediones (TZDs), a family of PPARgamma ligands, did not inhibit keratinocyte proliferation in normal murine skin. Here, we studied the effects of two TZDs, namely ciglitazone (10 mM) and troglitazone (1 mM), in the same murine model where epidermal hyperproliferation was reproduced by repeated barrier abrogation with tape stripping. Topical treatment with ciglitazone and troglitazone resulted in a marked and significant decrease in epidermal thickness. Furthermore, in all TZD-treated groups, we observed a significant decrease in keratinocyte proliferation using proliferating cell nuclear antigen, 5-bromo-2'-deoxyuridine, and tritiated thymidine incorporation. However, using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, we found no difference in apoptosis between different treatments, emphasizing that it is the antiproliferative role of these activators that accounts for the decrease of epidermal thickness. Finally, using immunohistochemical methods, we determined the effects of ciglitazone on keratinocyte differentiation in this hyperproliferative model. We observed an increased expression of involucrin and filaggrin following ciglitazone treatment, suggesting a pro-differentiating action of TZDs in this model. In summary, topical TZDs significantly reduce epidermal keratinocyte proliferation while promoting differentiation in a murine model of hyperproliferative epidermis. Together, these results suggest that in addition to their metabolic effects currently in use in the treatment of type 2 diabetes, topical TZDs could be considered as potential alternative therapeutic agents in hyperproliferative skin diseases such as psoriasis.     

An ex vivo human skin model for studying skin barrier repair

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32°C and 37°C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37°C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32°C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37°C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32°C and 37°C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37°C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32°C. Potentially, this model can be used for testing formulations for skin barrier repair.     

Establishment of a mouse skin model of the lichenification in human chronic eczematous dermatitis

BACKGROUND: Repeated mechanical stresses, such as scratching and rubbing, on a lesional skin area induce a rough skin condition known as lichenification in patients with chronic eczematous dermatitis. For ethical reasons, the pathomechanisms involved are difficult to study, so an animal model is required. OBJECTIVES: To study the pathomechanisms of the unique rough skin changes seen in chronic eczematous dermatitis, we established a mouse skin model by repeated tape stripping to inflict stratum corneum (SC) barrier disruption. The skin characteristics of the model were investigated biologically, histologically and pharmacologically. METHODS: Tape stripping was done on mouse back skin three times a week for 4 weeks. The skin changes were studied by obtaining negative replicas, haematoxylin and eosin staining, immunostaining for CD31 and BrdU, and measuring epidermal and cutaneous thickness and skin capacitance. Activities of matrix metalloproteinase (MMP)-2, 9 and urokinase-type plasminogen activator (uPA) in the skin tissues were analysed by zymography. The effects of MMP inhibitor and glycine were assessed. RESULTS: The repeated tape stripping produced crusting and desquamation at 48 h, followed 1 week later by the formation of shallow furrows, which became much deeper after 4 weeks, appearing as fine and regular wrinkles. The resultant wrinkled skin resembled lichenified skin seen in patients with chronic eczematous dermatitis. Histopathologically, we found acanthosis, hypergranulosis and hyperkeratosis even at 48 h, and the skin was 2.5 times thicker than untreated control skin at 4 weeks. We observed angiogenesis in the upper dermis at 1 and 4 weeks. Skin capacitance, a parameter of SC hydration, displayed consistently low levels throughout the experimental period. Although the dermis was also thickened, the activity of MMP-9 was sharply increased only at 24 and 48 h after tape stripping, declining thereafter to the control level. Topical applications of CGS-27023A (CGS), an MMP inhibitor, failed to suppress this tape-stripping-induced wrinkle formation. In contrast, topical applications of a barrier recovery accelerator, glycine, effectively inhibited the wrinkle formation induced by repeated tape stripping. CONCLUSIONS: The induction of fine and regular wrinkles by inflicting chronic SC barrier disruption in this model involves mainly epidermal changes, which is in sharp contrast to the mainly dermal changes induced by chronic ultraviolet B irradiation.     

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.     

Epidermal calcium release (ECR) in vivo sampled with a simple washout chamber technique

Background/aims: Epidermis forms the protective barrier of the skin by its outermost layer, stratum corneum. The purpose of this study was to investigate the epidermal barrier in view of epidermal calcium release (ECR), phosphate release, transepidermal water loss (TEWL) and skin surface pH. Calcium is mainly an intracellular ion. Calcium was sampled introducing a new and simple washout chamber technique for the study of epidermal release in vivo. Methods: Test sites on forearms of 13 healthy subjects were pre-treated with 24 h water occlusion, 24 h 2% sodium lauryl sulphate (SLS) or tape stripped. Both untreated and pre-treated test sites were exposed to a water washout chamber with 200µ deionized water as a solvent. Water washout chambers were removed after two hours and calcium and phosphate in the water was analyzed. Transepidermal water loss and pH were measured before and after the trial. Results: pH increased after tape stripping and after exposure to SLS. Transepidermal water loss increased significantly at all test sites. Calcium was significantly released from SLS-treated sites but not from tape stripped sites. There was generally a correlation between ECR, phosphate release, TEWL and pH. In this study ECR is showed to be a barrier marker of high reproducibility. Conclusions: Epidermal calcium release or ECR is found useful as an indicator of skin barrier function. Calcium release and increase of pH appear mainly to illustrate direct and corrosive damage to epidermal cells and functions contrasting TEWL, in this experiment probably reflecting intercellular damage of fracturing as exemplified by mechanical damage resulting from surface stripping. This new distinction of skin barrier damage into cellular damage resulting from a corrosive chemical trauma and intercellular damage and fracturing resulting from a mechanical trauma is exemplified in SLS provocative testing and tape stripping, the former characterized by increased ECR. The washout chamber technique was deemed technically reliable and reproducible, and has a major potential in experimental dermatology and skin pharmacology for the study of in vivo epidermal release of a range of endogenous and exogenous substances.     

Cannabinoid 1 receptors in keratinocytes attenuate fluorescein isothiocyanate‐induced mouse atopic‐like dermatitis

Atopic dermatitis is a chronic inflammatory disease characterized by an impaired epidermal barrier function combined with a chronic Th2‐type inflammatory response and an intense pruritus. Here, we used an experimental mouse model for Th2‐type contact hypersensitivity (CHS) to fluorescein isothiocyanate (FITC) to investigate the potential role of cannabinoid 1 receptors (CB1) in the pathophysiology of mouse atopic‐like dermatitis. Mice lacking CB1 receptors globally (Cnr1^?/?) or specifically in keratinocytes (KC‐Cnr1^?/?) as well as wild‐type (WT) control mice were sensitized and challenged with FITC. We examined ear swelling responses, transepidermal water loss, Th2‐type skin inflammatory responses and serum IgE levels. Both Cnr1^?/? and KC‐Cnr1^?/? showed enhanced CHS responses to FITC and a delayed epidermal barrier repair when compared with WT mice. mRNA levels for IL‐4, thymic stromal lymphopoietin (TSLP) and CCL8, as well as eosinophil activity, were significantly increased in inflamed ear tissue of FITC‐challenged Cnr1^?/? and KC‐Cnr1^?/? mice. Importantly, CB1 receptor‐deficient keratinocytes secreted increased levels of TSLP, a proinflammatory mediator that drives Th2‐type skin inflammation in atopic dermatitis, under basal and Th2‐type inflammatory conditions. Taken together, our results demonstrate that CB1 receptors in keratinocytes help to maintain epidermal barrier homoeostasis and attenuate Th2‐type allergic inflammatory responses. Based on our work, we propose that enhanced epidermal allergen penetrance cooperates with increased production of TSLP and CCL8 by epidermal keratinocytes for the induction of type 2 CD4+ T helper cells. Our results place keratinocytes at the cross‐roads of outside‐in and inside‐out pathophysiologic mechanisms of atopic dermatitis.     

Influx of calcium and chloride ions into epidermal keratinocytes regulates exocytosis of epidermal lamellar bodies and skin permeability barrier homeostasis

In the nervous system, influx of calcium and chloride ions into neurons regulates the signaling system by excitation and inhibition, respectively. In this study, we demonstrated the effects of the ion influx into epidermal keratinocytes in the permeability barrier repair process of the skin after damage. Topical application of the neurotransmitters glutamate and nicotine, which activate the calcium channel in neurons, delayed the barrier repair after tape stripping. In contrast, the neurotransmitters GABA and glycine, which activate the chloride channel in neurons, accelerated barrier repair. Topical application of the calcium ionophore ionomycin delayed barrier recovery and chloride ionophore 1 accelerated barrier repair after barrier disruption by tape stripping and acetone treatment. Ionomycin increased the intracellular calcium concentration in cultured keratinocytes whereas the chloride ionophore 1 increased the intracellular chloride ion concentration. In vivo light microscopy and electron microscopy observation showed acceleration of the exocytosis of lipid-containing lamellar bodies by the chloride ionophore and delay of the exocytosis by the calcium ionophore. These results suggest that, like the nervous system, influx of calcium and chloride ions into epidermal keratinocytes through ionotropic receptors plays a crucial role in cutaneous barrier homeostasis.     

Skin barrier response to occlusion of healthy and irritated skin: Differences in trans‐epidermal water loss,erythema and stratum corneum lipids

Background. Occlusion of the skin is a risk factor for development of irritant contact dermatitis. Occlusion may, however, have a positive effect on skin healing. No consensus on the effect of occlusion has been reached. Objectives. To investigate skin barrier response to occlusion on intact and damaged skin. Methods. In study A, the response to occlusion (nitrile glove material) for either 8 hr daily for 7 days or for 72 consecutive hours, respectively, was determined and compared with that of non‐occluded skin. In study B, the response to occlusion of for 72 consecutive hours of skin that had been damaged by either sodium lauryl sulfate (SLS) or tape stripping, respectively, was determined and compared with that of to non‐occluded pre‐damaged skin. Skin barrier function was assessed by measurements of trans‐epidermal water loss (TEWL) and erythema. In study A, stratum corneum lipids were analysed. Results. Occlusion of healthy skin did not significantly influence skin barrier function, ceramide profile or the ceramide/cholesterol ratio. Occlusion of the skin after SLS irritation resulted in higher TEWL than in the control (P = 0.049). Occlusion of the skin after tape stripping resulted in lower TEWL than in control skin (P = 0.007). Conclusions. A week of occlusion did not significantly affect healthy skin, but was found to decrease healing of SLS‐damaged skin, and to improve healing of tape‐stripped skin.     

Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold‐sensitive TRP receptors in epidermal permeability barrier homoeostasis

Please cite this paper as: Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold‐sensitive TRP receptors in epidermal permeability barrier homoeostasis. Experimental Dermatology 2010; 19 : 791–795. Abstract: TRPA1 and TRPM8 receptors are activated at low temperature (A1: below 17°C and M8: below 22°C). Recently, we observed that low temperature (below 22°C) induced elevation of intracellular calcium in keratinocytes. Moreover, we demonstrated that topical application of TRPA1 agonists accelerated the recovery of epidermal permeability barrier function after disruption. In this study, we examined the effect of topical application of TRPM8 modulators on epidermal permeability barrier homoeostasis. Immunohistochemical study and RT‐PCR confirmed the expression of TRPM8 or TRPM8‐like protein in epidermal keratinocytes. Topical application of TRPM8 agonists, menthol and WS 12 accelerated barrier recovery after tape stripping. The effect of WS12 was blocked by a non‐selective TRP antagonist, Ruthenium Red, and a TRPM8‐specific antagonist, BTCT. Topical application of WS12 also reduced epidermal proliferation associated with barrier disruption under low humidity, and this effect was blocked by BTCT. Our results indicate that TRPM8 or a closely related protein in epidermal keratinocytes plays a role in epidermal permeability barrier homoeostasis and epidermal proliferation after barrier insult.     

Acute cutaneous barrier disruption activates epidermal p44/42 and p38 mitogen-activated protein kinases in human and hairless guinea pig skin

Acute cutaneous barrier disruption of the skin elicits various homeostatic repair responses in the epidermis. Although several candidates for the signaling mechanisms that induce these responses have been reported, e.g. the calcium and ion concentration, peroxisome proliferator-activated receptor-alpha, and TNF-alpha signaling mediated by sphingomyelinases, the exact nature of the signals remains undertermined. Therefore, assuming that an important group of serine/threonine-signaling kinases, mitogen- and SAPK/JNK, might link the barrier disruption to the subsequent homeostatic responses, the activation of three MAPKs in hairless guinea pig or in human skin after barrier disruption was investigated. The epidermal barrier was insulated with tape stripping or organic solvents, and Western blotting, and immune complex kinase assay. In the skin of hairless guinea pigs, p44/42 MAPK and p38 MAPK, but nor SAPK/JNK, were continued to be activated for at least 180 min. The activation of p44/42 which positively correlated with the number of tape strippings, whereas K+ sucrose solution suppressed its activation. The activation of p44/42 MAPK was also induced by treatment of the skin with organic solvents. In similar fashion, p44/42 and p38 MAPKs were found to be activated in human skin after tape stripping. These results for strongly suggest that the activation of p44/42 and p38 MAPKs links the stimuli of barrier disruption to the subsequent homeostatic responses to repair the barrier defect.     

Inducible nitric oxide synthase is required for epidermal permeability barrier homeostasis in mice

Nitric oxide (NO) regulates a variety of epidermal functions, including epidermal proliferation, differentiation and cutaneous wound healing. However, whether nitric oxide (NO) and its synthetic enzymes regulate epidermal permeability barrier homeostasis is not clear. In the present study, we employed inducible nitric oxide synthase (iNOS) KO mice to explore the role of iNOS in epidermal permeability barrier homeostasis. Our results showed that iNOS mice displayed a comparable levels of basal transepidermal water loss rates, stratum corneum hydration and skin surface pH to their wild-type mice, but epidermal permeability barrier recovery was significantly delayed both 2 and 4 hours after acute barrier disruption by tape stripping. In parallel, expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes were lower in iNOS KO mice versus wild-type controls. Topical applications of two structurally unrelated NO donors to iNOS KO mice improved permeability barrier recovery kinetics and upregulated expression levels of mRNA for epidermal differentiation-related proteins and lipid synthetic enzymes. Together, these results indicate that iNOS and its product regulate epidermal permeability barrier homeostasis in mice.     

Transepidermal potassium ion, chloride ion, and water flux across delipidized and cellophane tape-stripped skin

The skin barrier was evaluated as a function of transepidermal water loss (TEWL) and electrolyte loss. Combination electrodes for chloride and pH determinations and a potassium ion electrode were utilized. Delipidization of the skin did not impair the electrolyte barrier, but did damage the epidermal water barrier. Cellophane tape stripping of normal stratum corneum resulted in an increase in outward transepidermal potassium and chloride ion flux, an increase in skin surface pH, and an increase in TEWL. It appears that damage to the epidermal water barrier does not necessarily result in damage to the epidermal electrolyte barrier. We found the potassium electrode facile to use and believe that a combination potassium electrode would be useful for investigating and assessing the epidermal electrolyte barrier.     

Topical hesperidin prevents glucocorticoid‐induced abnormalities in epidermal barrier function in murine skin

Systemic and topical glucocorticoids (GC) can cause significant adverse effects not only on the dermis, but also on epidermal structure and function. In epidermis, a striking GC‐induced alteration in permeability barrier function occurs that can be attributed to an inhibition of epidermal mitogenesis, differentiation and lipid production. As prior studies in normal hairless mice demonstrated that topical applications of a flavonoid ingredient found in citrus, hesperidin, improve epidermal barrier function by stimulating epidermal proliferation and differentiation, we assessed here whether its topical applications could prevent GC‐induced changes in epidermal function in murine skin and the basis for such effects. When hairless mice were co‐treated topically with GC and 2% hesperidin twice‐daily for 9 days, hesperidin co‐applications prevented the expected GC‐induced impairments of epidermal permeability barrier homoeostasis and stratum corneum (SC) acidification. These preventive effects could be attributed to a significant increase in filaggrin expression, enhanced epidermal β‐glucocerebrosidase activity and accelerated lamellar bilayer maturation, the last two likely attributable to a hesperidin‐induced reduction in stratum corneum pH. Furthermore, co‐applications of hesperidin with GC largely prevented the expected GC‐induced inhibition of epidermal proliferation. Finally, topical hesperidin increased epidermal glutathione reductase mRNA expression, which could counteract multiple functional negative effects of GC on epidermis. Together, these results show that topical hesperidin prevents GC‐induced epidermal side effects by divergent mechanisms.