State of the art in non‐invasive imaging of cutaneous melanoma

Background: This review focuses on looking at recent developments in the non‐invasive imaging of skin, in particular at how such imaging may be used at present or in the future to detect cutaneous melanoma. Methods: A MEDLINE search was performed for papers using imaging techniques to evaluate cutaneous melanoma, including melanoma metastasis. Results: Nine different techniques were found: dermoscopy, confocal laser scanning microscopy (including multiphoton microscopy), optical coherence tomography, high frequency ultrasound, positron emission tomography, magnetic resonance imaging, and Fourier, Raman, and photoacoustic spectroscopies. This review contrasts the effectiveness of these techniques when seeking to image melanomas in skin. Conclusions: Despite the variety of techniques available for detecting melanoma, there remains a critical need for a high‐resolution technique to answer the question of whether tumours have invaded through the basement membrane.     

Real‐time three‐dimensional imaging of epidermal splitting and removal by high‐definition optical coherence tomography

While real‐time 3‐D evaluation of human skin constructs is needed, only 2‐D non‐invasive imaging techniques are available. The aim of this paper is to evaluate the potential of high‐definition optical coherence tomography (HD‐OCT) for real‐time 3‐D assessment of the epidermal splitting and decellularization. Human skin samples were incubated with four different agents: Dispase II, NaCl 1 M, sodium dodecyl sulphate (SDS) and Triton X‐100. Epidermal splitting, dermo‐epidermal junction, acellularity and 3‐D architecture of dermal matrices were evaluated by High‐definition optical coherence tomography before and after incubation. Real‐time 3‐D HD‐OCT assessment was compared with 2‐D en face assessment by reflectance confocal microscopy (RCM). (Immuno) histopathology was used as control. HD‐OCT imaging allowed real‐time 3‐D visualization of the impact of selected agents on epidermal splitting, dermo‐epidermal junction, dermal architecture, vascular spaces and cellularity. RCM has a better resolution (1 μm) than HD‐OCT (3 μm), permitting differentiation of different collagen fibres, but HD‐OCT imaging has deeper penetration (570 μm) than RCM imaging (200 μm). Dispase II and NaCl treatments were found to be equally efficient in the removal of the epidermis from human split‐thickness skin allografts. However, a different epidermal splitting level at the dermo‐epidermal junction could be observed and confirmed by immunolabelling of collagen type IV and type VII. Epidermal splitting occurred at the level of the lamina densa with dispase II and above the lamina densa (in the lamina lucida) with NaCl. The 3‐D architecture of dermal papillae and dermis was more affected by Dispase II on HD‐OCT which corresponded with histopathologic (orcein staining) fragmentation of elastic fibres. With SDS treatment, the epidermal removal was incomplete as remnants of the epidermal basal cell layer remained attached to the basement membrane on the dermis. With Triton X‐100 treatment, the epidermis was not removed. In conclusion, HD‐OCT imaging permits real‐time 3‐D visualization of the impact of selected agents on human skin allografts.     

Two‐photon microscopy for intracutaneous imaging of stem cell activity in mice

The adult skin is a typical example of a highly regenerative tissue. Terminally differentiated keratinocytes are shed from the external layers of the epidermis or extruded from the skin as part of the growing hair shaft on a daily basis. These are effectively replenished through the activity of skin‐resident stem cells. Precise regulation of stem cell activity is critical for normal skin homoeostasis or wound healing and irregular stem cell proliferation or differentiation can lead to skin disease. The scarcity and dynamic nature of stem cells presents a major challenge for elucidating their mechanism of action. To address this, we have recently established a system for visualizing stem cell activity, in real time or long term, in the intact skin of live mice using two‐photon microscopy. The purpose of this review was to provide essential information to researchers who wish to incorporate two‐photon microscopy and live imaging into their experimental toolbox for studying aspects of skin and stem biology in the mouse model. We discuss fundamental principles of the method, instrumentation and basic experimental approaches to interrogate stem cell activity in the interfollicular epidermis and hair follicle.     

In vivo imaging reveals selective PPAR activity in the skin of peroxisome proliferator‐activated receptor responsive element‐luciferase reporter mice

Peroxisome proliferator‐activated receptors (PPARs) have been revealed as key regulators of several skin disorders. This has led to a growing interest in the development of drugs targeting PPARs as therapeutics for skin diseases. To evaluate skin PPAR activity, we developed peroxisome proliferator responsive element‐luciferase (PPRE‐Luc) mice, a mouse model in which the luciferase gene expression is under the control of a PPAR‐inducible promoter in all organs. Our aim was to define and validate experimental conditions to establish PPRE‐Luc mice as a valuable tool for in vivo non‐invasive evaluation of PPARs activation in the skin. We demonstrated by optical imaging that topical application of 40 mm of Luciferin for 10 min was enough to reveal the optimal luciferase activity in mice skin. The treatment of mice skin with the PPARγ and PPARα agonists, pioglitazone and WY14643, was associated with significant increase in photons emission reaching maximal signalling at 6 h. We have performed dose response studies by testing a large range of pioglitazone and WY14643 concentrations on mouse skin. The specificity of bioluminescence signal induced by pioglitazone and WY14643 was assessed using PPARγ and PPARα antagonists, GW9662 and GW6471, respectively. This approach revealed that the isoform specificity of PPARs agonists decreased when high ligand concentrations were applied on mouse skin. These results were further confirmed by in vitro measurement of luciferase activity in skin extracts. Overall, our results demonstrated that PPRE‐Luc mice represent a valuable reporter mouse model for the in vivo pharmacological profiling of drugs targeting PPARs in the skin.