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.     

Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis

Abstract:  Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.     

In vitro metabolism by human skin and fibroblasts of retinol, retinal and retinoic acid

The metabolism of radio-labelled retinol, retinal and retinoic acid by fresh human skin as well as by human dermal fibroblasts have been investigated in vitro . Surgically removed human skin biopsies were placed at the air-liquid interface, and treated topically for 24 h with retinoids. At the end of the treatment period, epidermis and dermis were separated by heat. Epidermis, dermis and medium were subsequently extracted and resulting fractions were analysed by HPLC. Dermal fibroblast cultures were treated and analysed in a comparable manner. Topical application of retinoids resulted in gradient concentrations within the skin. For each fraction, metabolites and unchanged product proportions were determined by HPLC. After treatment with retinol and retinal, low but significant amounts of retinoic acid were detected in the epidermis, as well as in the dermis (30 pmol to 90 pmol). In comparison, treatments with retinoic acid itself, led to higher level of retinoic acid in the epidermis and in the dermis (respectively 2050 and 420 pmol). Cultured human dermal fibroblasts, treated with retinol and retinal, formed retinoic acid as well as several other metabolites (retinol esters, reduction of retinal to retinol…). Taken together, our results are consistent with an action of retinol or retinal on the skin via a retinoic acid formation and a metabolic function of the dermis.     

Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy

Background/purpose: Multiphoton microscopy (MPM) enables the assessment of unstained living biological tissue with submicron resolution, whereas fluorescence lifetime imaging microscopy (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. The aim of this study was to compare the healthy skin of young individuals with that of older subjects, as well as to assess the skin at different body sites, by means of MPM and FLIM. Methods: Nineteen elderly patients were examined on the outer side of the forearm, whereas 30 young individuals were assessed on the dorsal and volar sides of the forearm and on the thigh. Results: Cell and nucleus diameters, cell density and FLIM vary according to the epidermal cell depth and the skin site. In elderly subjects, epidermal cells show morphologic alterations in shape and size, with smaller cell and nucleus diameters; the number of basal cells is decreased, whereas the mean fluorescence lifetimes at both the upper and the lower layers increase. Conclusion: This study provides quantitative and qualitative data on normal epidermis at different skin sites at different ages and represents a reference for the clinician attempting to understand the effectiveness of MPM and FLIM in discriminating diseased states of the skin from normal ones.     

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.     

Nonlinear spectral imaging of human hypertrophic scar based on two-photon excited fluorescence and second-harmonic generation

Background A noninvasive method using microscopy and spectroscopy for analysing the morphology of collagen and elastin and their biochemical variations in skin tissue will enable better understanding of the pathophysiology of hypertrophic scars and facilitate improved clinical management and treatment of this disease. Objective To obtain simultaneously microscopic images and spectra of collagen and elastin fibres in ex vivo skin tissues (normal skin and hypertrophic scar) using a nonlinear spectral imaging method, and to compare the morphological structure and spectral characteristics of collagen and elastin fibres in hypertrophic scar tissues with those of normal skin, to determine whether this approach has potential for in vivo assessment of the pathophysiology of human hypertrophic scars and for monitoring treatment responses as well as for tracking the process of development of hypertrophic scars in clinic. Methods Ex vivo human skin specimens obtained from six patients aged from 10 to 50 years old who were undergoing skin plastic surgery were examined. Five patients had hypertrophic scar lesions and one patient had no scar lesion before we obtained his skin specimen. A total of 30 tissue section samples of 30 μm thickness were analysed by the use of a nonlinear spectral imaging system consisting of a femtosecond excitation light source, a high‐throughput scanning inverted microscope, and a spectral imaging detection system. The high‐contrast and high‐resolution second harmonic generation (SHG) images of collagen and two‐photon excited fluorescence (TPEF) images of elastin fibres in hypertrophic scar tissues and normal skin were acquired using the extracting channel tool of the system. The emission spectra were analysed using the image‐guided spectral analysis method. The depth‐dependent decay constant of the SHG signal and the image texture characteristics of hypertrophic scar tissue and normal skin were used to quantitatively assess the amount, distribution and orientation of their collagen and elastin components. Results Our experiments and data analyses demonstrated apparent differences between hypertrophic scar tissue and normal skin in terms of their morphological structure and the spectral characteristics of collagen and elastin fibres. These differences can potentially be used to distinguish hypertrophic scar tissues from normal skin and to evaluate treatment responses. Conclusions All the measurements were performed in backscattering geometry and demonstrated that nonlinear spectral imaging has the ability to differentiate hypertrophic scar tissue from normal skin based on noninvasive SHG imaging, and TPEF imaging revealed the microstructure and spectral features of collagenand elastin fibres. With the advances in spectral imaging apparatus miniaturization, we have good reason to believe that this approach can become a valuable tool for the in vivo pathophysiology study of human skin hypertrophic scars and for assessing the treatment responses of this disfiguring disease in clinic. It can also be used to track the development of hypertrophic scars and to study wound healing processes in a noninvasive fashion without biopsy, fixation, sectioning and the use of exogenous dyes or stains.     

The evaluation of whitening efficacy of cosmetic products using a human skin pigmentation spot model

Background: To establish a pigmentation spot model on human skin and to assess whitening efficacy for whitening products by this established pigmentation spot model. Methods: Twenty subjects between 20 and 45 years old with skin phototype III or IV were selected. Three consecutive daily UV exposures were performed on buttocks of the subjects as follows: Day 1=1 minimal erythema dose (MED), Day 2=0.5 MED and Day 3=0.5 MED. After the first UV exposure, a selected whitening product was applied to the subjects twice a day on UV exposure area. The application of the whitening product to subjects on the exposed areas was continued till Day 27. CM2500d chromameter, Maxmeter MX18 and visual evaluation were used to assess changes of skin color. Results: A pigmentation spot model after UV exposure was established. The measurement of the pigment spot showed that value declined abruptly at Day 3 and then slowly reached to a lowest point at Day 6. value of the pigment spot almost remained at the same level until Day 20, thereafter increased slowly. The value showed an abrupt increase at Day 3 and slowly reached to a maximal level at Day 6. The value slowly declined toward its baseline level. Likewise, the erythema index also increased significantly at Day 3, and reached to a maximal level at Day 6 and then slowly declined. However, , and erythema indices did not return to their baseline levels during the 27‐day period of this study. On the other hand, value started to increase from Day 3 and such increase was observed continuously to Day 27. Melanin index also showed a slow increase during the first 3 days. It started to increase rapidly from Day 3 and a to maximal level at Day 9 and maintain at a plateau till Day 27 (with an exception at Day 13). To assess the whitening product by this pigmentation spot model, ΔL, , and ΔM values were analyzed. It showed that absolute ΔL value and value of whitening products were lower than those values of the vehicle of the whitening product at each checkpoint, while ΔM value of the whitening product was lower only at Day 9 and Day 20, although no statistically significant differences was found. The visual results also strongly supported that the whitening product enhanced the decrease of pigmentation. Conclusion: This study showed that repeated UV exposure was able to induce a long extensive period of pigment formation. The resulted pigmentation spot was able to maintain at an elevated level till Day 20. Clinical subjective evaluations together with combined objective instrument measurements were still important to assess whitening and spot‐removing ability of a material due to the instrument limitation for color differentiations. This kind of pigmentation spot model can be used to assess whitening efficacy for whitening or spot‐removing products. In addition, the combinations of subjective and objective methods were able to serve as advisable references to assess the whitening efficacy of products.     

In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography

Background: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography. Objectives: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed. Methods: Thirty healthy volunteers aged 21–82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth‐resolved intensity curves after correlation with multiphoton images. Results: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings. Conclusion: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science.     

In vivo dynamic thermal imaging of skin radiofrequency treatment

In recent years, popularity of radiofrequency (RF) has increased significantly. They are characterized by a low risk of complications and relatively high effectiveness. RF use high‐frequency currents causing oscillating motion of ions resulting in temperature rise stimulating skin regeneration processes. The aim of this work was the thermographic evaluation of the skin exposed to RF of different intensity. The dynamic thermal imaging was used to study the temperature of the skin exposed to RF. The research was carried out in two locations with different adipose tissue content: abdomen (ROI1) and forearm (ROI2). In the ROI1 area, RF was applied at nominal power range from 250 to 1750 W, while in ROI2 area: from 250 to 1000 W. The obtained thermographic data were fitted to exponential functions. A clear dependence of obtained thermokinetic parameters with the anatomical location of exposure to RF was demonstrated. Thicker layer of adipose tissue directly under the skin resulted in obtaining higher maximum temperatures of the skin surface during the procedure (maximum obtained temperature equaled 40.8°C). The temperature of the skin under the head of the device does not translate to subjective patient experiences. In anatomic locations filled with less adipose tissue mass, tolerance to RF is much lower. The dynamics of skin temperature changes, after the RF treatment, can be described by means of a single exponential function where the key parameter is the time constant t₁ defining the dynamics of skin temperature changes. The depth of the RF influence is slightly correlated with the RF power.     

Biophysical measurements of human forearm skin in vivo: effects of site, gender, chirality and time

Background/aims: Measurements of transepidermal water loss (TEWL) and skin colour are biophysical techniques commonly used to measure the in vivo skin effects of cosmetics, topical medicaments and chemical irritants. The purpose of this study was to investigate the variability of TEWL and skin colour on human forearm skin as a function of regional variation, gender and preferred chirality over an 8 h period.
Methods: Biophysical measurements of TEWL and skin colour were made at five sites on both forearms of male ( n =8) and female ( n =9) human volunteers in vivo (38% relative humidity, 21°C).
Results: Rates of TEWL at the forearm midpoint were 10% lower than at the forearm extremities ( P <0.01). Skin redness ( a *) near the wrist was 5–10% higher than at other sites ( P <0.05). Rates of TEWL were 5% higher in male volunteers ( P <0.05). Red and blue ( b *) colour measurements of male forearm skin differed by 18% and 20% in comparison with female, respectively. Rates of TEWL, skin brightness ( L *) and b * decreased by 9% ( P <0.05), 1.8% ( P <0.05) and 4% ( P <0.05), respectively, with time whereas a * and skin temperature increased by 4.5% ( P <0.01) and 7.2% ( P <0.01), respectively. There was a significant correlation between the change in all measured parameters with time.
Conclusions: Significant differences in TEWL and skin colour were identified that may have relevance in the design and interpretation of multivariate analyses of human forearm skin. Diurnal variation of TEWL, skin colour and temperature may have a single underlying mechanism.     

In vivo optical coherence tomography imaging of human skin: norm and pathology

Background/aims: Since the majority of skin diseases are known to be accompanied by structural alterations, research efforts are focused on the development of various novel diagnostic techniques capable of providing in vivo information on the skin structure. An essential parameter here is spatial resolution. In this paper we demonstrate the capabilities of optical coherence tomography (OCT) in detecting in vivo specific features of thin and thick skin. A particular focus is made on the identification of OCT patterns typical of certain pathological processes in skin, by performing parallel histological and tomographical studies. Methods: To obtain images of the skin, we used a compact fiber OCT system developed at the Institute of Applied Physics of the Russian Academy of Sciences. A low coherence source (superluminescent diode) operated at a wavelength of 1280 nm; the output power was 0.5–2 mW. This power is low enough to conform to the ANSI safety standards for light exposure. The in‐depth resolution limited by the spectral bandwidth (40–50 nm) of the probing light was ～20 μm. The lateral resolution determined by the probe light focusing ranged from 15 to 30 μm. In this series of experiments the maximum depth of imaging did not extend beyond 1.5 mm. Obtaining images of skin regions 2–6 mm long took 2–4 s. OCT capabilities for imaging normal skin of different localization and some skin diseases were studied in 12 healthy volunteers and 24 patients. Results: OCT imaging of the skin can detect in vivo such general pathological reactions of the human body as active inflammation and necrosis. OCT is useful for in vivo diagnosis of some specific processes in the skin, including hyperkeratosis, parakeratosis and formation of intradermal cavities. OCT imaging is noninvasive and therefore allows frequent multifocal examination of skin without any adverse effects. OCT can perform monitoring of disease progress and recovery in the course of therapy. Morphometric studies, measurements of the depth and extension of skin pathology within the human body can be easily performed by OCT. Conclusions: OCT allows imaging of subsurface soft tissues with the spatial resolution of 15–20 μm, a resolution one order of magnitude higher than that provided by other clinically available noninvasive diagnostic techniques. An imaging depth of up to 1.5–2 mm, given by current OCT technology, is sufficient to examine the skin. Real time OCT imaging can provide information not only on the structure, but also on some specific features in the functional state, of tissues. OCT imaging is a noninvasive technique, i.e., OCT does not cause trauma and has no side effects since it utilizes radiation in the near infrared wavelength range at a power as low as 1 mW.     

In vivo confocal laser scanning microscopy for non-invasive diagnosis of pemphigus foliaceus

Background/purpose: In vivo confocal laser scanning microscopy (CLSM) is a modern non-invasive method for investigation of the skin that allows real-time visualization of individual cells and sub-cellular structures at resolution similar to the one provided by routine histopathology. Our aim was to investigate the potential of CLSM for non-invasive diagnosis of pemphigus foliaceus (PF).
Methods: Pre-existing and mechanically induced lesions in two cases of PF were examined by means of CLSM, parallel to routine histology, direct immunofluorescence microscopy and enzyme-linked immunosorbent assay performed in the same patients.
Results: The morphological features characteristic for PF, namely an intraepidermal blister with acantholytic cells in the blister cavity, were readily detectable by means of CLSM. The findings were consistent in both patients and across the investigated lesions. The confocal images were consistent with the routine histology of the pre-existing lesions. No differences in the confocal images of pre-existing lesions compared with mechanically induced ones were observed.
Conclusions: Our findings suggest the potential of CLSM as a non-invasive tool for the diagnosis of pemphigus and differentiation of its subtype. Although at present the method cannot replace the current diagnostic standards for pemphigus, it may be successfully used as in vivo non-invasive screening tool to facilitate the diagnosis and point to the need for further investigation of the patient.     

Evaluation of effects of platelet-rich plasma on human facial skin

Abstract

Introduction: Platelet-rich plasma (PRP) has been used for rapid healing and tissue regeneration in many fields of medicine. This study was conducted to evaluate the effects of PRP application procedure on human facial skin. Methods: PRP was applied thrice at 2-week intervals on the face of ten healthy volunteers. It was applied to individual's forehead, malar area, and jaw by a dermaroller, and injected using a 27-gauge injector into the wrinkles of crow's feet. Participants were asked to grade on a scale from 0 to 5 for general appearance, skin firmness–sagging, wrinkle state and pigmentation disorder of their own face before each PRP procedure and 3 months after the last PRP procedure. While volunteers were evaluating their own face, they were also assessed by three different dermatologists at the same time by the same five-point scale. Results: There was statistically significant difference regarding the general appearance, skin firmness-sagging and wrinkle state according to the grading scale of the patients before and after three PRP applications. Whereas there was only statistically significant difference for the skin firmness–sagging according to the assessment of the dermatologists. Conclusion: PRP application could be considered as an effective procedure for facial skin rejuvenation.     

The effects of heating and cooling on ultraviolet radiation-induced erythema and pigmentation in human skin

Background/Purpose: As most biochemical systems are affected by temperature, thermal changes before or after ultraviolet (UV) irradiation could influence skin vascular blood flow changes and inflammatory responses. In this study, our aim was to investigate the influence of thermal changes on UV-induced acute skin reactions, namely, erythema and pigmentation.
Methods: Our volunteers consisted of 10 males, with ages ranging from 22 to 24 years and with Fitzpatrick's skin type III or IV. Skin temperatures were changed with a 45°C heating pad or by ice pack application before or after UV irradiation (control, 1 minimal erythema dose (MED), 2 MED) and then changes in erythema and pigmentation were measured by a Minolta Spectrophotometer CM-2002.
Results: The present study demonstrates that both heating and cooling increase skin erythema and reduce pigmentation, and that the timing of heating and cooling influences the UV-induced skin reaction. Pre-heating and post-cooling groups showed more UV-induced erythema than the post-heating and pre-cooling groups, respectively.
Conclusion: Our results indicate that alteration of skin surface temperature could modulate UV-induced erythema and pigmentation responses.     

Longitudinal in vivo tracking of adverse effects following topical steroid treatment

Topical steroids are known for their anti‐inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two‐photon excitation fluorescence microscopy (TPEF) of reduced nicotinamide adenine dinucleotide (NADH) to visualize the epidermal cell layers and second harmonic generation (SHG) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM) is also utilized to image intracellular NADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP)‐treated, mometasone furoate (MF)‐treated and untreated animals were imaged longitudinally using a custom‐built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime of NADH, suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment.     

Noninvasive methods for the assessment of photoageing

Although histopathological dermal elastosis is the current gold standard for the diagnosis of photoageing, noninvasive methods for quantifying the amount of photodamage to skin are clearly preferable. This study is the first to survey five noninvasive methods of assessing photoageing (clinical examination, spectrophotometry, skin surface topography, reflectance confocal microscopy and fluorescence lifetime imaging microscopy) in the same individual. Measurements for each noninvasive method were compared across nine individuals from three participant groups (‘younger’, ‘older’ and ‘photodamaged’) in UV‐protected volar and UV‐exposed dorsal forearm skin. Overall, participants in the younger group had the lowest measures of photodamage, while those in the photodamaged group had the highest, as indicated by each modality. The five noninvasive strategies surveyed in this study may demonstrate potential as a suitable methodology for the quantification of photoageing. The advantage of such noninvasive methods is that they allow for skin visualisation in vivo and repeated assessments of the same site. The main limitation of this study was its small sample size, which may have precluded many findings of statistical significance.