Mueller polarimetric imaging as a tool for detecting the effect of non-thermal plasma treatment on the skin.

Non-thermal plasma (NTP) is a promising technique studied for several medical applications such as wound healing or tumor reduction. The detection of microstructural variations in the skin is currently performed by histological methods, which are time-consuming and invasive. This study aims to show that full-field Mueller polarimetric imaging is suitable for fast and without-contact detection of skin microstructure modifications induced by plasma treatment. Defrosted pig skin is treated by NTP and analyzed by MPI within 30 minutes. NTP is shown to modify the linear phase retardance and the total depolarization. The tissue modifications are inhomogeneous and present distinct features at the center and the fringes of the plasma-treated area. According to control groups, tissue alterations are primarily caused by the local heating concomitant to plasma-skin interaction.

Cold Atmospheric Plasma Promotes Killing of Staphylococcus aureus by Macrophages.

Macrophages are important immune cells that are involved in the elimination of microbial pathogens. Following host invasion, macrophages are recruited to the site of infection, where they launch antimicrobial defense mechanisms. Effective microbial clearance by macrophages depends on phagocytosis and phagolysosomal killing mediated by oxidative burst, acidification, and degradative enzymes. However, some pathogenic microorganisms, including some drug-resistant bacteria, have evolved sophisticated mechanisms to prevent phagocytosis or escape intracellular degradation. Cold atmospheric plasma (CAP) is an emerging technology with promising bactericidal effects. Here, we investigated the effect of CAP on Staphylococcus aureus phagocytosis by RAW 264.7 macrophage-like cells. We demonstrate that CAP treatment increases intracellular concentrations of reactive oxygen species (ROS) and nitric oxide and promotes the elimination of both antibiotic-sensitive and antibiotic-resistant S. aureus by RAW 264.7 cells. This effect was inhibited by antioxidants indicating that the bactericidal effect of CAP was mediated by oxidative killing of intracellular bacteria. Furthermore, we show that CAP promotes the association of S. aureus to lysosomal-associated membrane protein 1 (LAMP-1)-positive phagosomes, in which bacteria are exposed to low pH and cathepsin D hydrolase. Taken together, our results provide the first evidence that CAP activates defense mechanisms of macrophages, ultimately leading to bacterial elimination. IMPORTANCE Staphylococcus aureus is the most frequent cause of skin and soft tissue infections. Treatment failures are increasingly common due to antibiotic resistance and the emergence of resistant strains. Macrophages participate in the first line of immune defense and are critical for coordinated defense against pathogenic bacteria. However, S. aureus has evolved sophisticated mechanisms to escape macrophage killing. In the quest to identify novel antimicrobial therapeutic approaches, we investigated the activity of cold atmospheric plasma (CAP) on macrophages infected with S. aureus. Here, we show that CAP treatment promotes macrophage ability to eliminate internalized bacteria. Importantly, CAP could trigger killing of both antibiotic-sensitive and antibiotic-resistant strains of S. aureus. While CAP did not affect the internalization capacity of macrophages, it increased oxidative-dependent bactericidal activity and promoted the formation of degradative phagosomes. Our study shows that CAP has beneficial effects on macrophage defense mechanisms and may potentially be useful in adjuvant antimicrobial therapies.

Physical plasma therapy accelerates wound re-epithelialisation and enhances extracellular matrix formation in cutaneous skin grafts.

Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full-thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full-thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal-epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD-dependent TGF-ß1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 °C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

Cold atmospheric plasma modulates endothelial nitric oxide synthase signalling and enhances burn wound neovascularisation.

Treatment with cold atmospheric plasma (CAP) has been reported to promote wound healing in animals. However, how this process is mediated remains unclear. In this study we examined the mechanisms which underlie the improved wound healing effects of CAP and the roles of associated reactive oxygen and nitrogen species (RONS), which are generated by plasma. By using in vitro models which mimicked various steps of angiogenesis, we demonstrated that CAP triggered the production of nitric oxide (NO), and enhanced cell migration and the assembly of endothelial cells into vessel-like structures. These are both hallmarks of the proliferative phase of wound healing. Using a mouse model of a third-degree burn wound, we went on to show that CAP treatment was associated with enhanced angiogenesis, characterised by accelerated in vivo wound healing and increased cellular proliferation. Here, CAP significantly increased the in vivo production of endothelial NO synthase (eNOS), an enzyme that catalyses NO synthesis in endothelial cells, and significantly increased the expression of pro-angiogenic PDGFRß and CD31 markers in mouse wounds. Mechanistically, we showed that CAP induced eNOS phosphorylation and activation, thereby increasing the levels of endogenous NO in endothelial cells. Increased NO generation facilitated by CAP further stimulated important pro-angiogenic VEGFA/VEGFR2 signalling in vitro. This proof-of-concept study may guide future efforts aimed at addressing the use of physical plasma and its therapeutic applications in a variety of pathological scenarios. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Attenuated total reflectance spectroscopy with chirped-pulse upconversion.

Chirped-pulse upconversion technique has been applied to attenuated total reflectance (ATR) infrared spectroscopy. An extremely broadband infrared pulse was sent to an ATR diamond prism and the reflected pulse was converted to the visible by using four-wave mixing in krypton gas. Absorption spectra of liquids in the range from 200 to 5500 cm(-1) were measured with a visible spectrometer on a single-shot basis. The system was applied to observe the dynamics of exchanging process of two solvents, water and acetone, which give clear vibrational spectral contrast. We observed that the exchange was finished within ∼ 10 ms.