大鼠皮肤角质层对荧光素钠脂质体透皮吸收的影响

目的 探讨大鼠皮肤角质层对荧光素钠脂质体透皮吸收的影响。方法 采用Scotch胶带剥脱皮肤角质层,利用荧光光度计、Franz扩散池和荧光显微镜研究皮肤角质层和活性层中荧光素钠的含量;观察皮肤剥脱角质层后荧光素钠脂质体透皮吸收能力的变化和荧光素钠在皮肤中的分布。结果 应用脂质体后皮肤各层荧光素钠的分布量均较相应溶液和凝胶作用后增加(P<0.01);荧光素钠在皮肤各层的分布比例发生了变化;剥脱角质层后脂质体荧光素钠混悬液和荧光素钠溶液各时间点累积透皮浓度相比较,差异无显著性(P>0.05);脂质体荧光素钠凝胶和荧光素钠凝胶相比较,各时间段累积透皮浓度差异无显著性(P>0.05)。结论 脂质体能增加皮肤各层的荧光素钠含量,并能够改变皮肤角质层和去角质层皮肤之间的荧光素钠比例。剥脱角质层后脂质体制剂透皮量显著增加,与相应的普通制剂相比差异无显著性;剥脱角质层后脂质体制剂的毛囊扩散途径不发生改变。     

MORPHOLOGY AND THICKNESS OF THE HUMAN STRATUM CORNEUM

SUMMARY.— The method of exhibiting the layers of cells of the stratum corneum described by Kligman and Christophers has been employed and elaborated, and has proved to be practical and reproducible. The cells can be counted and observations made on their morphology.
The shape of the cells treated by this method has been shown to be different at various sites and at different levels of the stratum corneum, and in pathological states.
The stratum corneum is probably more compact than has been hitherto supposed.
Differences in susceptibility to alkali have been demonstrated in the horny cells surrounding the eccrine sweat ducts and the pilosebaceous orifices.
There are also age differences in the susceptibility of the stratum corneum to alkali and possibly in its thickness.     

A new computer-based evaporimeter system for rapid and precise measurements of water diffusion through stratum corneum in vitro.

It is important to have reliable methods for evaluation of skin barrier function when questions such as barrier perturbing effects of different agents and occlusive effects of different formulations are to be elucidated. A wealth of clinical work relates to measurements of transepidermal water loss in vivo, a method much affected by ambient air relative humidity, temperature, skin irritation processes, psychologic status of the subject, etc., factors that cause the method to suffer from low precision (i.e., high random error). Relating to these obstacles, we have developed a closed in vitro system for measurements of water diffusion rate through pieces of isolated stratum corneum at steady-state conditions, where the relative humidity and temperature is held constant and data can be collected continuously. Our evaporimeter-based in vitro system has a more than 3-fold higher precision (lower random error) ( approximately 10%) than measurements of transepidermal water loss in vivo ( approximately 35%). The results of our study show that: (i) the corneocyte envelopes contribute to the barrier capacity of stratum corneum; (ii) removal of the lipid intercellular matrix results in approximately a 3-fold increase in the water diffusion rate through the isolated stratum corneum (n = 20; p < 0.05), not a 100-fold as has previously been suggested; (iii) exposure to sodium dodecyl sulfate in water does neither alter the water diffusion rate (n = 10; p > 0.05) nor the water holding capacity (n = 10; p > 0.05) of stratum corneum; (iv) exposure to 1 M CaCl2 in water yields an increased water diffusion rate through stratum corneum (n = 10; p < 0.05); and (v) when applied to the stratum corneum in excess concentrations, the penetration enhancer Azone has occlusive effects on water diffusion through the stratum corneum (n = 6; p < 0.05).     

Hydration characteristics of pathologic stratum corneum--evaluation of bound water

We measured in vitro the hygroscopicity and bound (non-freezing) water of various samples of pathologic horny layer obtained from the lesions of senile xerotic skin and psoriasis vulgaris and the normal horny layer from glabrous skin and plantar horny layer. The amount of water taken up by pathologic stratum corneum was much smaller than that by normal horny layer in an environment at a high relative humidity (RH). Tightly bound primary water to stratum corneum measured by Karl Fischer's method was about 5 mg/100 mg of dry stratum corneum in all the samples studied, while less tightly bound secondary water was much smaller in amount in pathologic stratum corneum than in the controls, i.e., 31.7 mg/100 mg dry scale from senile xerosis and 27.2 mg/100 mg dry psoriatic scale as compared with 38.2 mg/100 mg dry normal stratum corneum from glabrous skin and 37.3 mg/100 mg dry normal plantar stratum corneum. We believe that the low hygroscopicity of the pathologic stratum corneum is due to this smaller capacity for secondary bound water, which is responsible for the development of a dry scaly appearance even at high RH.     

There is no influence of a temperature rise on in vivo adsorption of UV filters into the stratum corneum

Temperature influences the stratum corneum adsorption of several topically applied compounds. This study was designed to evaluate the influence of the temperature on the stratum corneum adsorption of 3 UV filters. The UV filters were solubilized in two vehicles, an emulsion gel and petroleum jelly and applied at respectively, 31 and 40 degrees C during 30 min. In vivo stratum corneum UV filter content was measured using the tape stripping method. Similar amounts of UV filter were detected in the stratum corneum when comparing applications at the different temperatures. Application of the UV filters in the emulsion gel resulted in higher stratum corneum UV filter concentrations compared with application in the petroleum jelly. The application temperature did not influence the stratum corneum adsorption of the tested UV filters while the nature of the vehicle significantly influenced the amount of UV filters recovered from the stratum corneum.     

Measurement of the stratum corneum drug reservoir to predict the therapeutic efficacy of topical iododeoxyuridine for herpes simplex virus infection

A rapid, in vivo measurement of the penetration of antiviral compounds into the skin would improve our ability to predict the therapeutic efficacy of topical treatments for herpes simplex virus (HSV) infection. We have studied the concentration of iododeoxyuridine (IDU) in the stratum corneum of guinea pig skin by tape stripping at different time points after single and multiple topical doses of the drug. These results were correlated with the efficacy of topical IDU against an experimental cutaneous HSV infection. Ten adhesive tape strippings were performed on depilated guinea pig dorsum in vivo at serial intervals after a single topical dose of [3H]IDU. Iododeoxyuridine levels in the stratum corneum peaked at 1-3 h (67-70 mg/g of tissue) and then gradually declined over the next 3-24 h. We hypothesized that the peak IDU stratum corneum concentration would correlate with therapeutic efficacy. Accordingly, we determined the quantity of IDU in guinea pig stratum corneum 2 h after a topical application of seven different concentrations of IDU in dimethylsulfoxide (DMSO) and examined the in vivo efficacy of these formulations in an experimental dorsal cutaneous HSV-1 infection in guinea pigs. The results showed an excellent correlation between the quantity of IDU in the stratum corneum and reduction in lesion severity (r = 0.95-0.97). Fifteen percent IDU in DMSO provided the highest therapeutic efficacy (90-94%). We also studied the relationship between the clinical efficacy of different dosing frequencies and the amount of IDU in the stratum corneum. Serial IDU stratum corneum concentrations were measured over 24 h following 1, 2, 3, or 4 applications per day of 1, 3, and 15% IDU in DMSO treatments and parallel efficacy studies of the different regimens were conducted in the animal model. Within each dosing frequency, the cumulative amount of drug in the stratum corneum correlated with the strength of the test formulation and with efficacy in the animal model. For each of the three formulations, increasing the number of daily doses from one up to three led to progressive increases in cumulative stratum corneum IDU levels and clinical efficacy. An increase in the number of daily applications to four had little effect on drug efficacy and was associated with a plateau in stratum corneum IDU levels. Stratum corneum IDU concentrations were rapid and easy to determine and correlated well with clinical events.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of non-enzymatic glycosylation and heating on browning of human stratum corneum and nail.

The purpose of the present study was to examine the effect of non-enzymatic glycosylation and subsequent heating on the browning of the plantar stratum corneum and the finger-nail, and to elucidate the pathogenesis of the yellow skin and the yellow nail seen in diabetic subjects. We incubated stratum corneum and nail from non-diabetics in 0 (control), 10 (only nail), 20 (only nail), 100 and 250 mM glucose buffer at 37 degrees C for 5 days. These glycosylated samples were dialysed against distilled water for 96 h. Distilled water was changed every 24 h. Then samples were dried for 24 h. The extent of non-enzymatic glycosylation was measured by furosine content. Each 5 mg of sample was hydrolysed by 6 N HCl and processed for measurement of furosine by high-performance liquid chromatography. The rest of each sample was stored at 37, 42 (only nail), 47 and 52 degrees C for 14 days. Browning of the stratum corneum was assessed macroscopically, and that of the nail by spectrophotometry. Based on their spectrophotometric reflectances. Munsell's scores (H = hue score, V = lightness score, C = saturation score) and (H + C)/V were calculated for objective evaluation of browning. Incubation of the stratum corneum and nail with glucose buffer increased their non-enzymatic glycosylation (furosine) dose dependently. Macroscopically, the browning of the stratum corneum was enhanced in proportion to the glucose concentration and storage temperature. However, samples incubated in 10 and 20 mM glucose and stored at 42 degrees C did not show visible browning. Munsell's score of the nail samples treated by glycosylation and heating showed increased hue and saturation but reduced lightness. (H + C)/V values of these nail samples were significantly higher than those of the control. We could not detect any fluorescence with Wood light in the browned samples. The present in vitro study demonstrated that the browning of the stratum corneum and the nail depended on the extent of both non-enzymatic glycosylation and storage temperature. We suggested a hypothesis that the non-enzymatic glycosylation and the storage temperature of the stratum corneum and the nail might be a contributory factor in the development of yellow skin and yellow nail in diabetic patients.     

Intraepidermal neutrophils – a clue to dermatophytosis?

BACKGROUND: Dermatophyte infections are occasionally diagnosed by histopathology. Spongiotic or psoriasiform features are typical but non-specific, and neutrophils may be present within the stratum corneum. Traditionally, this latter finding has been felt to be a diagnostic clue for dermatophytosis, and usually precipitates a periodic acid Schiff (PAS-D) stain to confirm the presence of hyphae in the stratum corneum. Our objective was to evaluate whether the presence of neutrophils within the stratum corneum is a sensitive or specific test for dermatophytosis. METHODS: We performed a retrospective case-control study on 303 cases of spongiotic or psoriasiform dermatitides over a 35-month period. Hematoxylin and eosin (H&E) and PAS-D stains were utilized to identify intraepidermal neutrophils and fungi. RESULTS: The sensitivity and specificity for diagnosing dermatophyte infection based upon neutrophils within the stratum corneum were 62 and 59%, respectively. The positive and negative predictive values in our population were 4 and 98%, respectively. CONCLUSION: The histologic feature of neutrophils within the stratum corneum is neither sensitive nor specific in the diagnosis of dermatophytosis.     

A method for predicting steady-state rate of skin penetration in vivo

A simple in vivo method was proposed for predicting the steady-state rate of penetration of drugs across the stratum corneum. Both the diffusion coefficient and the partition coefficient in the stratum corneum can be determined by the amounts of drug in the stratum corneum at two time intervals under transient conditions after transdermal drug application. The amount of drug entering the stratum corneum is determined by 20 strippings with an adhesive tape. The steady-state rate of penetration was then calculated for the thickness of the stratum corneum and the concentration of the donor solution. The steady-state rates of penetration of ascorbic acid and estradiol across hairless mouse skin were evaluated from this in vivo approach and compared with those obtained from in vitro penetration experiment using excised hairless mouse skin. The data confirmed that the proposed in vivo method can predict the steady-state rate of penetration of these drugs across the stratum corneum in normal skin.     

By the grace of peeling: the brace function of the stratum corneum in the protection from photo-induced keratinocyte carcinogenesis

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG) is a safe and effective method for the rejuvenation of photo-damaged skin. The procedure removes photo-damaged stratum corneum, which consists of immature, fragile cornified envelopes (CEs) and stimulates the reconstruction of the stratum corneum with mature, rigid CEs. In UVB-irradiated hairless mice this procedure, which affects the stratum corneum only, suppresses skin tumor development. In addition, chemical peeling with SA-PAG suppresses p53 expression in mice and normalizes keratinocyte differentiation in both mice and humans. The stratum corneum functions as a barrier against physical and chemical insult and various infectious agents. Here, we hypothesize on a new function of the stratum corneum: a brace function that structurally protects keratinocytes from atypical differentiation or disordered proliferation. Although the precise mechanism remains to be elucidated, there is definite value to be gained from further investigation. This review discusses basic information about chemical peeling with SA-PEG, looks at its action on photo-induced tumor suppression, and proposes a new function for the stratum corneum in keratinocyte proliferation/differentiation.     

By the grace of peeling: the brace function of the stratum corneum in the protection from photo-induced keratinocyte carcinogenesis

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG) is a safe and effective method for the rejuvenation of photo-damaged skin. The procedure removes photo-damaged stratum corneum, which consists of immature, fragile cornified envelopes (CEs) and stimulates the reconstruction of the stratum corneum with mature, rigid CEs. In UVB-irradiated hairless mice this procedure, which affects the stratum corneum only, suppresses skin tumor development. In addition, chemical peeling with SA-PAG suppresses p53 expression in mice and normalizes keratinocyte differentiation in both mice and humans. The stratum corneum functions as a barrier against physical and chemical insult and various infectious agents. Here, we hypothesize on a new function of the stratum corneum: a brace function that structurally protects keratinocytes from atypical differentiation or disordered proliferation. Although the precise mechanism remains to be elucidated, there is definite value to be gained from further investigation. This review discusses basic information about chemical peeling with SA-PEG, looks at its action on photo-induced tumor suppression, and proposes a new function for the stratum corneum in keratinocyte proliferation/differentiation.     

The influence of stratum corneum hydration on body fat determination by bioelectrical impedance analysis

Background/aims: Bioelectrical impedance analysis (BIA) is used for the estimation of the amount of body fat. We evaluated the influence of the stratum corneum hydration at the contact areas used for BIA on the body fat estimation. Methods: Stratum corneum hydration was measured at the sole of the right foot and the palm of the right hand before and after contact with the Tanita Body Composition Analyzer TBF 410^® and the Omron Body Fat Analyzer^®, (n=128 females and 126 males), respectively. Changes in stratum corneum hydration during the contact time were calculated (ΔHYD). As a gold standard for body fat estimation, the underwater weighing method (UWW) was used and the deviation of this standard was calculated for the Tanita (DT) and the Omron (DO) measurement. Results: During contact with the Tanita, stratum corneum hydration increased significantly at the foot. Neither stratum corneum hydration measured at the respective contact sites before BIA nor ΔHYD at the respective skin sites was related to DT or with DO. Conclusion: The BIA measuring procedure using the Tanita instrument leads to an occlusive effect at the contact site. BIA for the determination of body composition is not influenced by stratum corneum hydration.     

Sebaceous gland secretion is a major physiologic route of vitamin E delivery to skin

Skin plays an important part in the protection against oxidative stressors, such as ultraviolet radiation, ozone, and chemicals. This study was based on the observation that upper facial stratum corneum contained significantly higher levels of the antioxidant alpha-tocopherol than corresponding layers of arm stratum corneum. We hypothesized that the underlying mechanism involves sebaceous gland secretion of vitamin E. To test this, we examined in eight human volunteers: (i) stratum corneum levels and distribution profiles of vitamin E in sites with a different sebaceous gland density (arm versus cheek); (ii) whether vitamin E is a significant constituent of human sebum; and (iii) if there is a correlation between levels of vitamin E and squalene, a marker of sebum secretion, in skin surface lipids. Using standardized techniques for stratum corneum tape stripping and sebum collection, followed by high-performance liquid chromatography analysis of tocopherols and squalene, we found that: (i) the ratio of cheek versus upper arm alpha-tocopherol levels was 20 : 1 for the upper stratum corneum and decreased gradually with stratum corneum depth; (ii) vitamin E (alpha- and gamma-tocopherol forms) is a significant constituent of human sebum and is continuously secreted at cheek and forehead sites during a test period of 135 min; and (iii) vitamin E correlates well with levels of cosecreted squalene (r2 = 0.86, p < 0.001). In conclusion, sebaceous gland secretion is a relevant physiologic pathway for the delivery of vitamin E to upper layers of facial skin. This mechanism may serve to protect skin surface lipids and the upper stratum corneum from harmful oxidation.     

Controlled removal of human stratum corneum by pulsed laser

A new method is presented for controlled removal of the stratum corneum of human skin. An excimer laser (193 nm wavelength, 14 ns pulsewidth) was used to remove stratum corneum from in vitro human skin samples by an ablative process. The tritiated water (3H2O) permeability constant and electrical resistance of skin samples were measured in a diffusion chamber apparatus to quantify the enhancement of skin permeability. Each laser pulse ablates about a micrometer of stratum corneum, which allows controlled removal of tissue. The maximum specific enhancement of the 3H2O permeability constant obtained after complete stratum corneum removal depends on the laser pulse energy used. The most gentle laser ablation, achieved with a radiant exposure of 70 mJ/cm2 per pulse, produced a 124-fold enhancement, which is comparable to that achieved after stratum corneum removal by tape-stripping or removal of epidermis by mild heat treatment. Rapid tissue ablation occurred at higher radiant exposures of 170-480 mJ/cm2 per pulse, but only a 45-fold enhancement of permeability was achieved. The precision with which stratum corneum can be ablated using excimer laser pulses may allow further basic research on the internal structure of stratum corneum and on the re-epithelization in controlled wounds. The technique may prove useful clinically to enhance percutaneous transport in applications such as topical delivery of drugs, patch testing, and percutaneous blood gas monitoring.     

Stratum corneum tryptic enzyme in normal epidermis: a missing link in the desquamation process?

Stratum corneum chymotryptic enzyme may be important in desquamation. It has also been suggested that other proteases, especially stratum corneum tryptic enzyme, may be involved. Stratum corneum tryptic enzyme has been purified and its cDNA has been cloned. Results from expression analyses indicate that stratum corneum tryptic enzyme is as skin specific as stratum corneum chymotryptic enzyme. In this work we have produced and characterized antibodies specific for stratum corneum tryptic enzyme. We have also by means of biochemical, immunochemical, and immunohistochemical methods performed studies on stratum corneum tryptic enzyme in normal human epidermis. Antibodies against bacterial recombinant stratum corneum tryptic enzyme were produced and purified by affinity chromatography. Two types of antibodies were obtained: one reacting only with pro-stratum corneum tryptic enzyme and one specific for the catalytically active part of stratum corneum tryptic enzyme. Immunohistochemistry with the antibodies reacting with pro-stratum corneum tryptic enzyme showed a staining pattern similar to stratum corneum chymotryptic enzyme-specific antibodies, i.e., the expression was confined to cornifying epithelia with a need of desquamation-like processes. Extracts of tape strips with superficial human stratum corneum were found to contain precursors as well as active forms of stratum corneum tryptic enzyme and stratum corneum chymotryptic enzyme. The enzymes had maximal activity at pH 8, but both had considerable activity also at pH 5.5. The results were compatible for a role of stratum corneum tryptic enzyme in desquamation. Stratum corneum tryptic enzyme may act in concert with stratum corneum chymotryptic enzyme and/or function as a stratum corneum chymotryptic enzyme-activating enzyme. The presence in normal superficial stratum corneum of precursors as well as of active forms of stratum corneum chymotryptic enzyme and stratum corneum tryptic enzyme, and the activity of both enzymes over a broad range of pH-values, suggest some possible ways by which the desquamation may be regulated.     

Skin barrier structure and function: the single gel phase model.

A new model for the structure and function of the mammalian skin barrier is postulated. It is proposed that the skin barrier, i.e., the intercellular lipid within the stratum corneum, exists as a single and coherent lamellar gel phase. This membrane structure is stabilized by the very particular lipid composition and lipid chain length distributions of the stratum corneum intercellular space and has virtually no phase boundaries. The intact, i.e., unperturbed, single and coherent lamellar gel phase is proposed to be mainly located at the lower half of stratum corneum. Further up, crystalline segregation and phase separation may occur as a result of the desquamation process. The single gel phase model differs significantly from earlier models in that it predicts that no phase separation, neither between liquid crystalline and gel phases nor between different crystalline phases with hexagonal and orthorhombic chain packing, respectively, is present in the unperturbed barrier structure. The new skin barrier model may explain: (i) the measured water permeability of stratum corneum; (ii) the particular lipid composition of the stratum corneum intercellular space; (iii) the absence of swelling of the stratum corneum intercellular lipid matrix upon hydration; and (iv) the simultaneous presence of hexagonal and orthorhombic hydrocarbon chain packing of the stratum corneum intercellular lipid matrix at physiologic temperatures. Further, the new model is consistent with skin barrier formation according to the membrane folding model of Norlén (2001). This new theoretical model could fully account for the extraordinary barrier capacity of mammalian skin and is hereafter referred to as the single gel phase model.     

Flow cytometric investigations of corneocytes from psoriatic scales and the effects of Ingram therapy

Flow cytometry has been used to study the stratum corneum. Following mechanical disruption and propidium iodide staining for DNA, analysis of the fluorescence intensity profile permits discrimination between nucleated and anucleate corneocytes. It was established that the nucleated corneocyte from parakeratotic stratum corneum contains double-stranded DNA in the normal diploid amount. A model investigation is described following the normalization of the psoriatic stratum corneum during Ingram therapy. It is shown that the initially high percentage of nucleated corneocytes (50%) falls exponentially during the first weeks of therapy. The rate of normalization seems to offer a quantitative and objective approach for evaluation and optimization of therapy.     

On the use of infrared spectroscopy for the in vivo measurement of the water content of the horny layer after application of dermatologic ointments

Summary Infrared-spectroscopic investigations of the water content of the stratum corneum after application of emulsions and wash solutions are reported. As a methodologic variation infrared spectroscopy is applied after stripping the skin to determine the hydration of various depths of the stratum corneum by ointments. Using soap as an example, a method of correcting the overlap effects between the ointment and skin spectrums is presented which expands the application possibilities of infrared spectroscopy. An o/w emulsion with plenty of water and a w/o emulsion containing less water produce to almost the same degree a hydration of the deeper layers of the stratum corneum which lasts for at least 40 min. The report in the literature that soap solutions produce a stronger hydration of the stratum corneum than surfactant solutions is not supported.Supported by the Deutsche Forschungsgemeinschaft (Grant Gl 91/4)