Sodium fluorescein (Fl-Na), Nile Red, 10% formalin solution, Mayer's hematoxylin solution and Eosin Y were obtained from Fuji Film Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Trypsin from bovine pancreas and trypsin inhibitor from Glycine max (soybean) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Glass capillaries (φ = 1.5 mm) were obtained from Toho Co., Ltd. (Tokyo, Japan).
SMS2-KO mice with a C57BL/6 background were generated by homologous recombination using targeted vectors. The mice were kept under a 12-h light/dark cycle in a conventional animal room maintained at 22 ± 2°C and 55 ± 5% humidity. A total of 28 SMS-KO mice and 24 WT mice (Seven-to-nine-week-old male and female) were used for all experiments. All procedures were approved by the Ethics Committee of Josai University (approval Nos. 2013-4, 2017-7), and all methods were implemented in accordance with Josai University's relevant animal care and use guidelines and regulations (approval Nos. H25089, JU18095). In addition, P1A diffusion prevention measures were used in all experiments. All experimental were performed in accordance with the ARRIVE guidelines (http://arriveguidelines.org).
Hematoxylin eosin (HE) staining.
The skin was collected, cut into 5 ´ 10 mm pieces, placed in a plastic dish and embedded with the O.C.T. (optimal cutting temperature) compound (Sakura Finetech Japan Co., Ltd., Tokyo, Japan). Frozen tissue sections 10 µm thick were prepared by using a Leica CM3050 S cryostat (Leica Biosystems, Nußloch, Germany) at in-house temperature (CT) of − 25°C and sample table temperature (OT) of − 25°C. Thin sliced sections were immersed in a 10% formalin solution for 30 min and then immersed in the order of 100, 90, 80 and 70% ethanol for hydrophilic treatment. After washing with water, the sections were immersed in Mayer's hematoxylin solution for 6 min and washed with water. The sections were then immersed in Eosin Y solution containing acetic acid. For dehydration treatment, thin sliced sections were soaked in the order of 70, 80, 90 and 100% ethanol and then soaked in xylene for clearing. The sections were observed with an all-in-one fluorescence microscope (BZ-X710, Keyence Corporation, Osaka, Japan).
In vitro permeation study.
Full-thickness skin or stripped skin by the tape stripping technique was collected from WT and SMS2-KO mice. The modified Franz cells (effective permeation area: 1.77 cm2) were mounted with the epidermis on the donor side and the dermis on the receiver side. One-and-a-half milliliters 1 mM Fl-Na aqueous solution or 1 mM Nile Red in methanol was placed on the donor side, and 4.5 mL of PBS (–) solution or 50% methanol solution was placed on the receiver side. Receiver solutions were taken at 1, 2, 3, 4, 5 and 6 h of full thickness skin, or 10, 20, 30, 40, 50 and 60 min of stripped skin. The amount of Fl-Na (Ex: 495 nm/Em: 520 nm) or Nile Red (Ex: 553 nm/Em: 637 nm) that had permeated samples were measured using a microplate reader. The cumulative amount of permeation and the skin permeation rate (flux) were calculated.
Preparation of stratum corneum.
The mouse skin was peeled off, immersed in a PBS (–) solution containing 0.1% trypsin and incubated at 4°C for 21 h. The skin sample was then immersed at 37°C for a further 3 h. The reaction was stopped by immersing the skin sample in a PBS (–) solution of 0.1% trypsin inhibitor for 5 min. The SC was collected, washed in pure water and dried. The SC was stored in an environment where the humidity was controlled by a saturated aqueous solution of potassium sulfate (room temperature, relative humidity 97.6 ± 0.6%).
Small- and wide-angle X-ray scattering.
X-ray scattering experiments on the SC were carried out using the beam line BL40B2 (Structural Biology II) at a large synchrotron radiation facility (SPring-8, Sayocho, Hyogo, Japan). The SC samples were filled in glass capillaries, and small- and wide-angle X-ray scattering were measured simultaneously. An imaging plate was used as the detector. The measurement conditions were set as follows: reference material, silver behenate (AgBh); wavelength, 0.08 nm; X-ray energy, 15 keV; camera length, 550 mm; and exposure time, 30 s. The obtained intensity profile was plotted against the scattering vector S (nm− 1) in the small-angle and wide-angle regions, where S = (2/λ) sin θ, λ is the X-ray wavelength and 2θ is the scattering angle. The observed wide-angle peaks were analyzed by fitting to a Gaussian function and a straight line. The peak intensity was calculated based on the analyzed results.
The X-ray scattering peaks of the hydrocarbon chain packing structure of intercellular lipids in the SC appeared at S = 2.4 nm− 1 (0.42 nm) in the hexagonal and orthorhombic structures and at S = 2.7 nm− 1 (0.37 nm) in the orthorhombic structure. The integrated intensity ratio (R0.42/0.37) of the 0.41-nm peak and the 0.37-nm peak was calculated from the intensities obtained above. Further, the sample temperature was increased from 10 to 120°C at a rate of 2°C/min, and during heating the wide-angle X-ray scattering experiment was performed. The measurement conditions in this experiment were set as: reference material, silver behenate; wavelength, 0.07 nm; camera length, 2195 mm; exposure time, 5 s.
Data are shown as the mean and standard deviation (mean ± S.D.) and statistically processed using JMP Pro version 13.0 (SAS Institute, Cary, NC, USA). For statistical tests, the Student's t-test was used after confirming equal variance by the F-test.