Dead Sea therapy is one of the oldest forms of treatment for skin disease and some chronic inflammatory diseases like arthritis and psoriasis. Much of the research to date has attributed the clinical effects of Dead Sea therapy to its mineral composition; mostly to magnesium salts. Magnesium salts, such as magnesium sulphate (Epsom salts), have long been used as a spa product and as a therapeutic to manage clinical conditions. The rationale of this PhD research was to understand the role of topically applied magnesium ions in epidermal integrity of human skin. We compartmentalized this research project into studying physiological, metabolic and proteolytic changes occurring at different layers of the skin. To begin with, we investigated the permeability of magnesium ions through stratum corneum. In Chapter 3, we developed a method to stain skin sections using mag-fura-2, a fluorescent dye that specifically binds to magnesium ions. When magnesium chloride (MgCl2) solution was topically applied on normal and tape stripped skin, we observed that it is possible for magnesium ions to permeate through stratum corneum. We also varied the concentration and time of exposure of MgCl2 solutions and found that at higher magnesium concentration, permeability is faster and penetration increases with progression of time. To further characterize permeability of the skin to magnesium, we blocked individual hair follicles using a novel method and subsequently treated skin with MgCl2 solution. We found that hair follicles significantly contribute towards the permeation of magnesium ions. In Chapter 4, the hypothesis tested was that magnesium accelerates barrier recovery. Experiments were conducted on volunteers who did not have any history of skin conditions. We caused stratum corneum disruption on their volar forearm through tape stripping and subsequently treated that defined region with different salt solutions. The parameters we considered for understanding barrier function were transepidermal water loss, skin hydration and skin pH over 96 hours with repeated treatment at 24-hour intervals. We found that treatment with a variety of salt solutions, including magnesium chloride did not aid in lowering the TEWL to levels pre-tape stripping and no changes to skin pH were observed. However, we observed that treating the skin with 1.9 M magnesium solution nearly 2 fold (p < 0.05) increased hydration at 6 hours, relative to baseline at time zero, whereas water treatment increased hydration only 1.2 fold, and the hydration returned to normal levels within 24 hours in both cases. Skin barrier formation is a continuous process and occurs as a result of differentiating epidermal cells subsequently leading to terminal differentiation. In the case of disrupted barrier, the epidermal cells differentiate to replenish the lost layers of stratum corneum. Therefore as an extension to Chapter 4, experiments in Chapter 5 involved understanding the metabolic changes occurring in all three layers of the epidermis – granulosum, spinosum, basale. We used multiphoton tomography coupled with fluorescent lifetime imaging microscopy to observe volunteers 0, 24 and 96 hours. Defined regions of volar forearm were treated with water or 1.9M MgCl2 solution on normal and tape stripped skin at 24-hour intervals over 96 hours. In all the three layers, we observed that redox ratio significantly decreased (p<0.05) immediately after tape stripping and significantly increased (p<0.05) after 24 hours in the magnesium treated site in granulosum and spinosum layer, when compared to the normal skin site. This increase is indicative of changes in metabolic activity in the granular layer, suggestive of signs of differentiation and cell proliferation. However, we did not observe any significant change in NAD(P)H/FAD lifetime nor in the ratio of nuclear area to total cellular area (N/C).
In order to gain more understanding about the role of magnesium in terminal differentiation, as reported in Chapter 6 we studied the filaggrin (FLG) regulation under varying concentrations of salt solutions and due to stimulation by external factors. We found that treating the excised skin with high concentrations of magnesium and calcium chloride solutions resulted in increased levels of FLG after 30 minutes of treatment. The increase in FLG levels could be due to the interaction of divalent ions with the binding sites present in the profilaggrin, subsequently exposing the sites for cleavage by enzymes such as caspase-14. Also, inflicting damage to the epidermis in excised skin using 2,4-dinitrochlorobenzene (DNCB) and tape stripping increased FLG levels when incubated for 20 hours. Also, we found that treating the skin with these salt solutions prior to stimulating them with DNCB or TS did not allow FLG levels to peak above those observed with unstimulated skin. Increased levels of FLG as a result of salt treatment could have a possible role in preparing the epidermal cells for terminal differentiation eventually leading to barrier formation. We applied salt solution treatments on the volar forearm of human volunteers to measure physiological parameters and found a significant increase in hydration, but not skin pH when compared to the control site. This ensured that the changes observed in FLG levels are not due to pH changes.
From these results we conclude that magnesium ions can penetrate through healthy skin with intact stratum corneum, with significant contribution from hair follicles. At high magnesium concentrations the permeation of Mg2+ into the epidermis increases with time and is significantly higher than the baseline concentration of Mg2+ in the epidermis of untreated controls after 15 minutes of exposure. Even though magnesium treatment caused an increase in skin hydration at 6 hours, we did not find effects on TEWL or skin pH. Magnesium treatment also increased the redox ratio of cells in granulosum and spinosum layer indicating changed metabolic activity. We also found that magnesium ions could be involved in proteolytic cleavage of profilaggrin preparing the epidermal cells for terminal differentiation and providing a possible preventive effect against external stimulants.