SKIN PROTECTION 83


“exposed” to the ML pollutants mix by vaporisation for 90 minutes whereas control were not exposed. After exposure, all explants were


returned to an incubator under standard culture conditions for 24 hours. On D0, the 3 explants from the batch T0 were collected and cut in two parts. Half was fixed in buffered formalin solution and half was frozen at -80°C. On D5, 24 hours after the end of pollutant exposure, 3 explants from the concerned batches were collected and treated in the same way than on D0. After fixation for 24 hours in buffered formalin, the samples were dehydrated and impregnated with paraffin using a Leica PEARL dehydration automat. Samples were then embedded using a Leica EG 1160 embedding station and 5μm thick sections were made using a Leica RM 2125 Minot- type microtome, and sections mounted on Superfrost®


histological glass slides.


Microscopic observations were performed using a Leica DMLB or Olympus BX43 microscope. Images were digitised with a numeric DP72 Olympus camera with CellD storing software. Cell viability of epidermal and dermal structures was assessed after Masson’s trichrome staining, Goldner variant.21


Immuno-histochemistry and microscopy MMP-1 (matrix metalloproteinase-1) immunostaining was performed on FFPE skin sections with a polyclonal anti-MMP1 antibody (Sigma, ref. M4696) for 60 minutes at room temperature, and a biotin conjugated secondary antibody. As for Nrf2, staining was revealed using HRP- avidin/biotin complex (Vector Vectastain RTU Universal), and a violet substrate of peroxidase (VIP, Vector laboratories, SK4600). The immunostaining was performed manually and assessed by microscopic observations. Nrf2 (oxidative stress transcription


factor) immunostaining was performed on formol-fixed paraffin embedded (FFPE)


Figure 3: Simplified schematic of the effects of reactive oxygen species (ROS) generated by pollution. Under oxidative stress, Nrf2 is activated, translocated to the nucleus, with an ensuing antioxidant cascade of events. NfkB stimulated by ROS causes an induction of inflammation by various intermediaries and the expression of matrix metalloproteinases resulting in tissue matrix degradation.


skin sections and stained with monoclonal anti-Nrf2 antibody (Abcam, ref. ab76026, clone EP1809Y), for 60 minutes at room temperature, and a biotin conjugated secondary antibody. Staining was revealed using HRP-avidin/biotin complex (Vector Vectastain RTU Universal) and a violet substrate of peroxidase (VIP, Vector laboratories, SK4600). Immunostaining was performed using an automated slide processing system (Autostainer, Dako), and assessed by microscopic observations. Cell viability remained good during the


experimental process. Any minor epidermal alterations were within normal parameters of the study protocol (data not


shown). Exposing skin explants with the standardised pollutant mix resulted in effects in both Nrf2 expression and MMP-1 expression.


Protective effects against MMP-1 Expression At the end of the treatment phase (5 days), the effects of MXSO were clear in the pollution induced over-expression of MMP- 1, whereby MXSO reduced its expression as compared to control samples. Under normal conditions, MMP-1 is an interstitial Type I collagenase responsible for the breakdown of collagens Type I and Type III in the skin. One aspect of reactive oxygen


September 2020


PERSONAL CARE EUROPE


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