Lars Norlén

Cryo-electron microscopy of vitreous sections

Since the beginning of the 1980s, cryo‐electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3‐d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo‐electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3‐d reconstruction.

The Human Skin Barrier Is Organized as Stacked Bilayers of Fully Extended Ceramides with Cholesterol Molecules Associated with the Ceramide Sphingoid Moiety

The skin barrier is fundamental to terrestrial life and its evolution; it upholds homeostasis and protects against the environment. Skin barrier capacity is controlled by lipids that fill the extracellular space of the skins surface layer--the stratum corneum. Here we report on the determination of the molecular organization of the skins lipid matrix in situ, in its near-native state, using a methodological approach combining very high magnification cryo-electron microscopy (EM) of vitreous skin section defocus series, molecular modeling, and EM simulation. The lipids are organized in an arrangement not previously described in a biological system-stacked bilayers of fully extended ceramides (CERs) with cholesterol molecules associated with the CER sphingoid moiety. This arrangement rationalizes the skins low permeability toward water and toward hydrophilic and lipophilic substances, as well as the skin barriers robustness toward hydration and dehydration, environmental temperature and pressure changes, stretching, compression, bending, and shearing.

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

Abstract The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

Stratum corneum swelling. Biophysical and computer assisted quantitative assessments.

Abstract The aim of this study was to characterize the swelling behaviour of the stratum corneum. Stratum corneum pieces isolated from the breast region of 20 different females were incubated in distilled water at two different temperatures (20° C and 45° C) for 90 min and 24 h, respectively. Half of the stratum corneum pieces were previously extracted with chloroformmethanol (2 : 1). The area-enlargement was photographically recorded. The thickness enlargement was determined using a confocal laser scanning microscope. The average swelling (99% confidence interval) in the area dimension at 20° C was 8.4% ± 1.4% ( n = 20), which corresponded to an average swelling in the length (lateral) dimension of approximately 4.1%. The swelling in the thickness dimension was 26.3% ± 16.3% ( n = 8). The swelling was most pronounced in the thickness dimension and was complete after 90 min of water immersion ( P < 0.01, n = 5). In addition, the removal of the intercellular lipids with chloroform/methanol (2 : 1) induced a decreased swelling in the samples ( P < 0.01, n = 20). An increase in temperature of the water from 20° C to 45° C resulted in an increase in swelling ( P < 0.01, n = 20). Taken together our results support the idea that the mechanism of stratum corneum swelling is linked to the intercellular lipid structure and hence to skin barrier function.

A novel approach to the understanding of human skin barrier function

The basis for externally caused skin disorders is penetration of the skin barrier. A recent model for the skin barrier, the domain mosaic model, based on current knowledge of the physics of lipid bilayer organization gave tentative explanations for several aspects of function. It is demonstrated here that a development of the model explains how the requirements are met for a water-tight structure that will still allow a controlled, minute loss of water, the perspiratio insensibilis, necessary for maintaining plasticity of the keratin. A major advantage of the extended model is that it allows an interpretation of the changes imposed on the structure when in contact with detergents and/or penetration enhancers.

Molecular cryo‐electron tomography of vitreous tissue sections: current challenges

Electron tomography of vitreous tissue sections (tissue TOVIS) allows the study of the three‐dimensional structure of molecular complexes in a near‐native cellular context. Its usage is, however, limited by an unfortunate combination of noisy and incomplete data, by a technically demanding sample preparation procedure, and by a disposition for specimen degradation during data collection. Here we outline some major challenges as experienced from the application of TOVIS to human skin. We further consider a number of practical measures as well as theoretical approaches for its future development.

Electrical impedance and other physical parameters as related to lipid content of human stratum corneum.

Background/aims: In previous studies we have shown that variations in the properties of the stratum corneum are reflected by alterations in electrical impedance. The aim of this study was to explore the ability of the electrical impedance technique to detect changes in the lipid content of the stratum corneum, and to compare It with the other non‐invasive methods, measurement of transepidermal water loss and of skin moisture.

Structure-related aspects on water diffusivity in fatty acid–soap and skin lipid model systems

Simplified skin barrier models are necessary to get a first hand understanding of the very complex morphology and physical properties of the human skin barrier. In addition, it is of great importance to construct relevant models that will allow for rational testing of barrier perturbing/occlusive effects of a large variety of substances. The primary objective of this work was to study the effect of lipid morphology on water permeation through various lipid mixtures (i.e., partly neutralised free fatty acids, as well as a skin lipid model mixture). In addition, the effects of incorporating Azone((R)) (1-dodecyl-azacycloheptan-2-one) into the skin lipid model mixture was studied. Small- and wide-angle X-ray diffraction was used for structure determinations. It is concluded that: (a) the water flux through a crystalline fatty acid-sodium soap-water mixture (s) is statistically significantly higher than the water flux through the corresponding lamellar (L(alpha)) and reversed hexagonal (H(II)) liquid crystalline phases, which do not differ between themselves; (b) the water flux through mixtures of L(alpha)/s decreases statistically significantly with increasing relative amounts of lamellar (L(alpha)) liquid crystalline phase; (c) the addition of Azone((R)) to a skin lipid model system induces a reduction in water flux. However, further studies are needed to more closely characterise the structural basis for the occlusive effects of Azone((R)) on water flux.

Skin Lamellar Bodies are not Discrete Vesicles but Part of a Tubuloreticular Network.

Improved knowledge of the topology of lamellar bodies is a prerequisite for a molecular-level understanding of skin barrier formation, which in turn may provide clues as to the underlying causes of barrier-deficient skin disease. The aim of this study was to examine the key question of continuity vs. discreteness of the lamellar body system using 3 highly specialized and complementary 3-dimensional (3D) electron microscopy methodologies; tomography of vitreous sections (TOVIS), freeze-substitution serial section electron tomography (FS-SET), and focused ion beam scanning electron microscopy (FIB-SEM) tomography. We present here direct evidence that lamellar bodies are not discrete vesicles, but are part of a tubuloreticular membrane network filling out the cytoplasm and being continuous with the plasma membrane of stratum granulosum cells. This implies that skin barrier formation could be regarded as a membrane folding/unfolding process, but not as a lamellar body fusion process.

Molecular Structure and Function of the Skin Barrier

A novel experimental approach combining high-resolution cryoelectron microscopy of vitreous tissue section (CEMOVIS) defocus series with electron microscopy simulation was employed to show that the stratum corneum lipid matrix is organized as stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety. The new knowledge may serve as a molecular platform for in silico approaches to identify molecules for enhancing skin penetration for percutaneous drug delivery as well as for tightening a leaking barrier in dry skin conditions.