In vivo morphological characterisation of skin by MRI micro-imaging methods

BACKGROUND/PURPOSE: Quantitative assessments in skin layers using images obtained with standard magnetic resonance imaging (MRI) sequences are limited, since the stratum corneum and dermis, the layers of most clinical interest, have low signal due to their short spin-spin relaxation, T2. METHODS: In the present work, different methods of MRI contrast, such as magnetisation transfer contrast (MTC), T1-weighting (where T1 is spin-lattice relaxation time), T2*-weighting (where T2* is the combination of T2 and magnetic field in-homogeneity effect) and chemical shift, were used. These techniques were combined with high-resolution MRI. RESULTS: We found that skin is a very MT active tissue, and MTC provides data enabling the evaluation of how the tissue in skin layers interacts with the interstitial fluids. Details obtained from high-resolution high-quality in vivo skin images with different contrast allowed for differentiation of skin layers, sub-layers and excellent correlation of MR data with known histological features and water constituent of skin layers. CONCLUSION: Combining MT and other MRI data employing other contrast mechanisms provides a superior non-invasive in vivo technique for visualisation and also quantitative assessment of the constituents of the stratum corneum, epidermis, papillary dermis, reticular dermis and hypodermis as major structural layers of the skin. This type of study can be extended to cutaneous disease states or skin ageing, where defects in water mobility, concentration and/or macromolecular structural changes are expected.     

Anatomy and physiology of subcutaneous adipose tissue by in vivo magnetic resonance imaging and spectroscopy: Relationships with sex and presence of cellulite

Background Little is still known concerning subcutaneous adipose tissue and cellulite, and controversial questions are still under discussion.
Aims Magnetic resonance imaging and spectroscopy were used to address two unresolved questions relating to the anatomy and physiology of subcutaneous adipose tissue.
Methods Using high spatial resolution magnetic resonance imaging we characterized the topography of the dermo- hypodermal junction, and the three-dimensional architecture of the subcutaneous fibrous septae. Using proton spectroscopy, we measured water and lipid fractions within a fat lobule, and T₁ and T₂ values of the detected compounds. All these data were analysed according to sex and presence of cellulite.
Results MR imaging quantified deeper indentations of adipose tissue into the dermis, and evidenced for the first time a great increase in the thickness of the inner fat layer in women with cellulite. Moreover, 3D reconstruction of the fibrous septae network showed a higher percentage of septae in a direction perpendicular to the skin surface in women with cellulite; but our study also depicted the tortuous aspect of this network. MR proton spectroscopy could not show any differences related to sex or presence of cellulite concerning T₁ and T₂ relaxation times of the detected compounds within a fat lobule, neither the unsaturated lipid fraction, the saturated lipid fraction, nor the water fraction.
Conclusions Magnetic resonance imaging showed that the 3D architecture of fibrous septae couldn't be modelled simply as perpendicular planes for women and tilted planes at 45° for men. MR spectroscopy did not confirm the hypothesis of increased water content in the adipose tissue of women with cellulite as suggested by others, except if such water would be located in the connective septae.     

Pilot study of dermal and subcutaneous fat structures by MRI in individuals who differ in gender, BMI, and cellulite grading

BACKGROUND/AIMS: Puckered, dimply skin on the thighs, hips, and buttocks is known as cellulite. The cause of cellulite is not known, although there are a number of different hypotheses. In this study, we use magnetic resonance (MR) micro-imaging to study cellulite skin. To the best of our knowledge, this is the first reported MR study of cellulite. METHODS: High-resolution in vivo MR images of the postlateral thigh skin of two male groups and four female groups were obtained. Subjects were grouped according to their body mass index (BMI) and cellulite grade. A qualitative assessment of how MRI can be used to differentiate skin tissue at different levels of cellulite grading was performed. RESULTS: We found that changes in skin architecture with cellulite can be visualized by in vivo MR micro-imaging. The skin fat layers beneath the dermis and down to the level of muscles are well visualized in the images. Also, the diffuse pattern of extrusion of underlying adipose tissue into dermis is clearly imaged, and was found to correlate with cellulite grading. We also show that other skin tissue parameters such as (a) the percentile of adipose vs. connective tissue in a given volume of hypodermis and (b) the percentile of hypodermic invaginations inside the dermis are correlated with cellulite grade. CONCLUSION: MR images can be interpreted to measure tissue parameters correlated with cellulite. Considering that we had only three subjects in each group, the achievements of this pilot study were highly satisfactory. We have shown that the in vivo micro-MR is a technique able to detect the effects of cellulite and gender. This study can be extended for further investigations of drugs and/or medical devices for cellulite treatment.     

Die kutane Mikrodialyse Anwendung in der Dermatologie

The determination of concentrations of exogenous or endogenous substances in the dermis, e.g. for pharmacokinetic studies, is technically difficult. Performing skin biopsies or inducing suction blisters results in the disintegration of the tissue and allows only single measurements. Recently, cutaneous microdialysis, a new simple and minimally invasive technique for continuously measuring of substances in the dermis in vivo, has been introduced in dermatological research. According to the principle of dialysis, a semipermeable membrane is inserted in the dermis and due to a concentration gradient between the interstitial space and a perfusate, substances diffuse through the pores of the membrane and can be analyzed in the dialysate. Cutaneous microdialysis represents a useful technique for pharmacokinetic or pharmacodynamic studies as well as for investigations regarding cutaneous physiology and pathophysiology.     

Magnetic resonance chemical shift microimaging of aging human skin in vivo: initial findings

Background/aims: In recent decades, interest has increased in the chronological and environmental factors governing the aging of skin. Various methods have been used for determining water and lipid content of human skin as a function of subject age. Magnetic resonance chemical shift imaging (CSI) offers a noninvasive technique for observing detailed distributions of water, lipids and other chemicals in the skin, and thus may be useful in dermatogerontology. Methods: Human skin was examined in vivo on nine healthy volunteers, both male and female. Localized ¹H spectra of the skin were obtained from voxels 78 μm thick and parallel to the skin surface. Unique water and lipid profiles were observed for different individuals, showing the composition and microstructure of epidermis, dermis and hypodermis in each subject. These allowed the quantification of skin thickness in vivo by the first appearance of triacylglyceride olefinic protons at the dermal-hy-podermal junction, or alternatively by the degree of lipid infiltration into dermis. Results: The relative concentration of free water in the skin, normalized to skin thickness, was observed to be slightly greater in older subjects and also in tanned subjects. More significantly, a microstructural feature common to every subject, i.e., the position of a possible capillary plexus in the dermis, showed migration toward the skin surface with advancing age. Conclusions: Such observations are consistent with previous studies of skin aging by other techniques and show promise for CSI in dermatogerontology as a non-invasive means for determination of skin water in vivo.     

In vivo characterization of minipig skin as a model for dermatological research using multiphoton microscopy

Minipig skin is one of the most widely used non-rodent animal skin models for dermatological research. A thorough characterization of minipig skin is essential for gaining deeper understanding of its structural and functional similarities with human skin. In this study, three-dimensional (3-D) in vivo images of minipig skin was obtained non-invasively using a multimodal optical imaging system capable of acquiring two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy (FLIM) images simultaneously. The images of the structural features of different layers of the minipig skin were qualitatively and quantitatively compared with those of human skin. Label-free imaging of skin was possible due to the endogenous fluorescence and optical properties of various components in the skin such as keratin, nicotinamide adenine dinucleotide phosphate (NAD(P)H), melanin, elastin and collagen. This study demonstrates the capability of optical biopsy techniques, such as TPEF and FLIM, for in vivo non-invasive characterization of cellular and functional features of minipig skin, and the optical image-based similarities of this commonly utilized model of human skin. These optical imaging techniques have the potential to become promising tools in dermatological research for developing a better understanding of animal skin models, and for aiding in translational pre-clinical to clinical studies.     

Age and skin structure and function, a quantitative approach (I): blood flow, pH, thickness, and ultrasound echogenicity

BACKGROUND/PURPOSE: The aging process has been studied with fervor recently, given our shifting demographics. Since age's effects are so manifest in skin's appearance, structure, mechanics, and barrier function, it is not surprising that much effort has been placed in research to better understand them. Quantitative measurements permitted by bioengineering have allowed us to objectively and precisely study aging skin. These overviews piece together the immense amounts of information that have emerged from recent technological advances in dermatological research in order to develop a unified understanding of the quantitative effects of age on the skin. METHODS: We performed a literature on age-related changes in blood flow, pH, skin thickness, and ultrasound imaging data, searching Pub-med, Em-Base, Science Citation Index, and the UCSF dermatological library's collection of books on the topic of aging skin. RESULTS: Despite the many tools and techniques available for quantitative analysis of skin, age studies are often conflicting, especially in the areas of blood flow and skin thickness. Trends indicate that blood flow may decrease with age, especially in sites exposed to the environment. pH apparently varies little until the age of 70, after which it declines. Skin thickness data are difficult to interpret; while the stratum corneum is generally accepted to maintain its thickness during aging, dermal, epidermal, and whole skin thickness changes are controversial. Ultrasound reveals the appearance of a subepidermal low echogenic band that thickens with age, especially in environmentally exposed areas. Some studies also indicate the presence of an echogenic band in the lower dermis which thins with increased age. However, the whole dermis appears to become more echogenic in elderly people. CONCLUSION: Much remains to be done if we are to reach consensus on the effects of age on skin structure and function. Future studies would be benefited by increased standardization of skin sites tested, methodology, and increased sample size.     

In vivo proton relaxation times analysis of the skin layers by magnetic resonance imaging

If in vivo magnetic resonance imaging is nowadays a powerful non-invasive method in medical diagnosis, its application in order to study the skin in vivo is not yet in common use because skin imaging requires a high resolution, at least in the direction perpendicular to the skin surface. We have therefore designed a specific imaging module, which, connected to a standard whole-body imager at 1.5 Tesla, allows us to obtain in vivo magnetic resonance images of skin on most parts of the body. With a depth resolution of about 70 microns, we are able to differentiate the skin layers: epidermis, dermis, subcutaneous fat, and even a thickened stratum corneum on palm as well as on heel. This paper reports the T1 and T2 water proton relaxation times of the different skin layers, in vivo, which are magnetic resonance parameters extracted from the images. Results show that skin layers are characterized by shorter T2 relaxation times than other biologic soft tissues. On the contrary, the measured T1 values are in the same range as in other tissues. These short T2 values may be assigned to the fibrous protein content of the skin and particularly of the dermis. This study on normal skin is the precursor of further works such as the influence of aging. As regards skin pathologies, it will be a powerful tool to follow the evolution of skin diseases under treatment.     

Penetration of lysophosphatidylcholine into the dermis.

The ability of Lysophosphatidylcholine (LPC) to penetrate into the dermis and its degradation were investigated in vivo using hairless rats. Phosphatidylcholine (PC) was used as a control. Radioactively labelled LPC and PC were applied on the surface of skin, and the radioactivities of the epidermis and dermis were measured at 8, 24, and 48 hours. The recoveries of radioactive materials of LPC and PC within the area of epidermis and dermis at 8 hours were 0.21% and 0.25%; at 24 hours, 0.68% and 0.31%; and at 48 hours, 0.42% and 0.92%. No radioactivity was detected in serum. The radioactive substances which had penetrated were identified as LPC, PC, diglycerides, and free fatty acids. It was also found that topical application of LPC did not change the structure of skin as seen by microscopical examination. These findings indicate that a small amount of LPC can penetrate without the damaging skin structure and is enzymatically degraded into several lipids. Since LPC has bactericidal and antiviral activity, this substance could be an useful agent for dermatological use.     

Fluorescence fibre-optic confocal microscopy of skin in vivo: microscope and fluorophores

BACKGROUND/AIMS: Fibre-optic confocal imaging in vivo is a new approach in the assessment of human skin. The objective is to describe a novel instrument and its operation and use in combination with fluorophores. METHODS: The Stratum is a fibre-optic fluorescence confocal microscope especially developed for the study of skin and mucous membranes. The system is flexible and any body site can be studied with a hand-held scanner. The light source is a 488 nm argon ion laser. Horizontal (en face) images of the epidermis and outer dermis are produced with cellular resolution. Magnification is approximately 1000 x . Fluorescein sodium is routinely used as fluorophore (intradermal injection or application to the skin surface). This fluorophore is safe for human use in vivo, but other substances (rhodamine B, Acridine Orange, green fluorescent protein, curcumin) have also been studied. RESULTS: The instrument produces sharp images of epidermal cell layers from the epidermal surface to the sub-papillary dermis, with sub-cellular resolution. The scanner is flexible in use. The technique of intradermal fluorophore injection requires some skill. CONCLUSIONS: We consider this fibre-optic instrument a potentially important tool in skin research for non-invasive optical biopsy of primarily the epidermis. Present use is focussed on research applications, where the fluorophore distribution in the skin may illustrate morphological changes in the epidermis.     

Prediction of chemical absorption into and through the skin from cosmetic and dermatological formulations

Background  To date, risk assessment following topical exposure to cosmetic/dermatological formulations cannot be precisely evaluated.
Objectives  To provide a tool for optimization of active permeation into/through skin and for risk assessment.
Methods  A predictive model was developed for estimating the cumulative mass of a chemical absorbed into and across the skin from a cosmetic/dermatological formulation. Account was taken of (i) the ionization state of the chemical, to correct the skin/vehicle partition coefficient; and (ii) the nature of the cosmetic/dermatological formulation. Three specific assumptions were made: firstly, steady-state transport across the skin was achieved despite application of a finite dose of chemical; secondly, vehicle effects were small relative to the precision of the prediction; and, thirdly, each formulation could be treated as an oil-in-water emulsion, in which only that fraction of the chemical in the aqueous phase was available to partition into the stratum corneum. A database of 101 ex vivo human skin experiments involving 36 chemicals was analysed.
Results  For 91% of the data, the difference between predicted and experimental values was less than a factor 5; when the aforementioned corrections were not used, on the other hand, only 26% of the data was well predicted. The model was successfully applied to predict skin absorption of two compounds not included in the database, for which in vitro percutaneous penetration from cosmetic vehicles have been measured.
Conclusion  A model has been developed to predict the mass of a chemical absorbed into and through the skin from a cosmetic or dermatological formulation.     

In vivo quantification of epidermis pigmentation and dermis papilla density with reflectance confocal microscopy: variations with age and skin phototype

Reflectance confocal microscopy (RCM) may help to quantify variations of skin pigmentation induced by different stimuli such as UV radiation or therapeutic intervention. The objective of our work was to identify RCM parameters able to quantify in vivo dermis papilla density and epidermis pigmentation potentially applicable in clinical studies. The study included 111 healthy female volunteers with phototypes I-VI. Photo-exposed and photo-protected anatomical sites were imaged. The effect of age was also assessed. Four epidermis components were specifically investigated: stratum corneum, stratum spinosum, basal epidermal layer and dermo-epidermal junction. Laser power, diameter of corneocytes and upper spinous keratinocytes, brightness of upper spinous and interpapillary spinous keratinocytes, number of dermal papillae and papillary contrast were systematically assessed. Papillary contrast measured at the dermo-epidermal junction appeared to be a reliable marker of epidermis pigmentation and showed a strong correlation with skin pigmentation assessed clinically using the Fitzpatrick's classification. Brightness of upper spinous and interpapillary spinous keratinocytes was not influenced by the skin phototype. The number of dermal papillae was significantly lower in subjects with phototypes I-II as compared with darker skin subjects. A dramatic reduction in the number of dermal papillae was noticed with age, particularly in subjects with fair skin. The method presented here provides a new in vivo investigation tool for quantification of dermis papilla density and epidermal pigmentation. Papillary contrast measured at the dermo-epidermal junction may be selected as a marker of skin pigmentation for evaluation in clinical studies.     

Topical L-thyroxine: The Cinderella among hormones waiting to dance on the floor of dermatological therapy?

Topical hormone therapy with natural or synthetic ligands of nuclear hormone receptors such as glucocorticoids, vitamin D analogues and retinoids has a long and highly successful tradition in dermatology. Yet the dermatological potential of thyroid hormone receptor (TR) agonists has been widely ignored, despite abundant clinical, cell and molecular biology, mouse in vivo, and human skin and hair follicle organ culture data documenting a role of TR-mediated signalling in skin physiology and pathology. Here, we review this evidence, with emphasis on wound healing and hair growth, and specifically highlight the therapeutic potential of repurposing topical L-thyroxine (T4) for selected applications in future dermatological therapy. We underscore the known systemic safety and efficacy profile of T4 in clinical medicine, and the well-documented impact of thyroid hormones on, for example, human epidermal and hair follicle physiology, hair follicle epithelial stem cells and pigmentation, keratin expression, mitochondrial energy metabolism and wound healing. On this background, we argue that short-term topical T4 treatment deserves careful further preclinical and clinical exploration for repurposing as a low-cost, effective and widely available dermatotherapeutic, namely in the management of skin ulcers and telogen effluvium, and that its predictable adverse effects are well-manageable.     

Magnetic resonance imaging of the skin

A thorough examination of the skin is essential to screen various diseases accurately, evaluate the effectiveness of topically applied drugs and assess the results of dermatological surgeries such as skin grafts. The assessment of skin properties is also crucial in the cosmetics industry, where it is important to evaluate the effects skin care products have on these properties. The simplest and most widely used method of skin evaluation, the ‘naked eye’ assessment, enables researchers to assess only the skin surface and involves a large amount of inter‐observer variability. Thanks to a great progress that has been made in physics, electronics and computer engineering in recent years, sophisticated imaging methods are increasingly available in day‐to‐day studies. The aim of this review was to present one of these techniques, namely the magnetic resonance imaging (MRI), and to discuss its possible use in skin examination and analysis. We present basic principles of MRI, as well as several interesting applications in the field of dermatology, and discuss the advantages and limitations of this method.     

共聚焦激光扫描显微镜在皮肤科的应用

共聚焦激光扫描显微镜是一种新兴的非侵入性图像分析仪器，具有高分辨率实时成像的特点。它可以观察活体内和体外的皮肤图像而不需要常规的组织固定、切片、染色。该技术在皮肤科研究中的应用及其临床意义日益得到重视。对于皮肤病的诊断和治疗，共聚焦激光扫描显微镜是非常有前景的技术，它为皮肤科学的发展开辟了广阔前景，促进了皮肤病学的医学研究和临床治疗。     

Sensitive skin: correlation with skin surface microrelief appearance

BACKGROUND/PURPOSE: Sensitive skin is a condition associated with reduced tolerance to environmental factors and/or the application of topical products, such as cosmetics. Its pathophysiology has not been fully elucidated and few data are available on its prevalence. The aim of this study was to investigate possible correlation between objective sensitivity and skin surface microrelief. METHODS: During an epidemiological survey conducted for a campaign promoted by International Society of Plastic Dermatology in Italy, 243 adult healthy subjects of both sexes with no evident dermatological disorder but positive to the lactic acid stinging test, were submitted to cyanoacrylate stratum corneum stripping from the volar forearm for the determination of the irregularity of the skin surface microrelief (irregularity skin index (ISI)). RESULTS: A significant correlation was found between intensity of symptoms in stingers and ISI (r(s)=-0.47; P<0.001). CONCLUSION: Sensitive skin is common in the healthy population. ISI can contribute towards the identification of subjects with sensitive skin and the development of more specific skin treatments for this prevalent condition.     

Protective effects of astaxanthin on skin: Recent scientific evidence,possible mechanisms,and potential indications

Astaxanthin is a naturally occurring ketocarotenoid which has been found to have numerous biological functions, with its strong antioxidant property being the prominent feature. The compound has attracted a great amount of interest with respect to its potential utilization in the betterment of human health. In the recent past, astaxanthin has been extensively studied with respect to its possible effect on skin health, with positive results. Astaxanthin has also shown to have anti‐inflammatory, immune‐modulating, and DNA repair properties, which have further encouraged its usage to maintain skin health and tackle skin damage. In this review article, we highlight the pharmacokinetic profile of the antioxidant in brief and describe the findings of various recent published research articles which studied the effect of astaxanthin in improvement of skin health. We also mention the possible mechanisms which form the basis of the positive dermatological effects of astaxanthin and the potential indications of the antioxidant molecule in cosmetology and dermatology.     

Mineralocorticoid receptor blockade improves glucocorticoid‐induced skin atrophy but partially ameliorates anti‐inflammatory actions in an irritative model in human skin explants

We recently demonstrated that blockade of the mineralocorticoid receptor (MR) effectively ameliorated GC‐induced skin atrophy in healthy human skin explants and epidermal MR knockout mice. However, whether MR blockade improves the therapeutic index of glucocorticoids (GCs) in skin pathology was not investigated. We assessed the effects of GCs, MR antagonists (MRA) or both, in SDS‐treated human skin explants. All treatments restored SDS‐augmented epidermal thickness but only GC plus MRA restored the expression of COL1A1. However, MRA alone or in combination with GCs may exert a dual role in regulating inflammatory cytokines. Thus, although combined treatment may be beneficial to improve irritative skin, extensive in vivo testing is required to establish whether the anti‐inflammatory effects of GCs are maintained in the presence of MRA.     

The Kollias legacy: Skin autofluorescence and beyond

In a paper published at the J Invest Dermatol in 1998 Nik Kollias and coworkers described distinct changes in skin native fluorescence associated with skin aging and photoaging, using in vivo fluorescence excitation spectroscopy. The assignment of the 295 nm band to tryptophan fluorescence had a profound significance influencing many later studies from multiple groups. The reproducible changes in skin native fluorescence suggested that aging causes predictable alterations in both the epidermis and the dermis, whereas chronic UV exposure induces the appearance of new fluorophores. This seminal, but insufficiently widely appreciated work deserves re‐examination as it points to important horizons in future experimental dermatology, such as cancer diagnostics, diabetes, wound healing, and understanding skin aging and photoaging mechanisms.     

Infrared radiation-induced matrix metalloproteinase in human skin: implications for protection

Human skin is exposed to infrared radiation (IR) from natural and artificial sources. In previous studies, near IR radiation (IRA; 760-1,440 nm) was shown to elicit a retrograde mitochondrial signaling response leading to induction of matrix metalloproteinase-1 (MMP-1) expression. These studies, however, have exclusively employed cultured human skin fibroblasts ex vivo. Here, we have assessed the in vivo relevance of these observations by exposing healthy human skin in vivo to physiologically relevant doses of IRA. Eighty percent of the tested individuals responded to IRA radiation by upregulating of MMP-1 expression. Specifically, IRA irradiation caused increased expression of MMP-1 in the dermis, but not in the epidermis. Raman spectroscopy revealed that IRA radiation also caused a significant decrease in the antioxidant content of human skin. In vitro studies had previously shown that IRA-induced MMP-1 expression was mediated through an oxidative stress response, which originates from the mitochondrial electron transport chain. We now report that incubation of cultured human dermal fibroblasts or treatment of human skin with specific antioxidants prevented IRA radiation-induced MMP-1 expression in vitro and in vivo. Thus, IRA irradiation most likely promotes premature skin aging and topical application of appropriate antioxidants represents an effective photoprotective strategy.