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New fluorescent three-dimensional and deep-imaging technique confirms a direct relationship between the acrosyringium and vesicles/pustules of palmoplantar pustulosis

      Palmoplantar pustulosis (PPP) vesicles arise from eccrine sweat in the acrosyringium and subsequently develop into pustulovesicles and pustules [
      • Murakami M.
      • Ohtake T.
      • Horibe Y.
      • et al.
      Acrosyringium is the main site of the vesicle/pustule formation in palmoplantar pustulosis.
      ,
      • Murakami M.
      • Terui T.
      Palmoplantar pustulosis: current understanding of disease definition and pathomechanism.
      ]. However, it is very difficult to determine whether a PPP vesicle lies within or around an acrosyringium because of the technical limitations of routine histopathological examination. Recently, we developed a high-quality fluorescence imaging system using transparency-enhancing technology [
      • Mizutani H.
      • Ono S.
      • Ushiku T.
      • et al.
      Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: a porcine model.
      ] and an improved fluorescent solvatochromic probe that changes emission spectra and fluorescent intensity depending on the hydrophobicity of lipid surrounding the probe [
      • Valanciunaite J.
      • Kempf E.
      • Seki H.
      • et al.
      Polarity mapping of cells and embryos by improved fluorescent solvatochromic pyrene probe.
      ]. In other words, fluorescent solvatochromic probe that varies its fluorescence color (wavelength) and intensity depending on its location between plasma and intracellular membranes. This new technique makes it possible to obtain high-quality, three-dimensional (3D) epidermal images, particularly of acrosyringia [
      • Murakami M.
      • Kawakami R.
      • Niko Y.
      • et al.
      High-quality fluorescence imaging of the human acrosyringium using a transparency: enhancing technique and an improved, fluorescent solvatochromic pyrene probe.
      ]. We obtained 3D images of PPP vesicles to directly explore the associations between PPP vesicles/pustules and the acrosyringium. We evaluated six biopsy specimens obtained from patients at Ehime University Hospital diagnosed with PPP based on their clinical features, histopathologies, and disease courses (supplement table) [
      • Teymouri M.
      • Halabchi F.
      • Mirshahi M.
      • et al.
      Comparison of plantar pressure distribution between three different shoes and three common movements in futsal.
      ]. All procedures were approved by the Ethics Committee of Ehime University, and the study adhered to all relevant principles of the Declaration of Helsinki. The sample preparation procedure prior to fluorescence microscopy has been described previously [
      • Murakami M.
      • Kawakami R.
      • Niko Y.
      • et al.
      High-quality fluorescence imaging of the human acrosyringium using a transparency: enhancing technique and an improved, fluorescent solvatochromic pyrene probe.
      ]. Briefly, punch biopsy samples 2 mm in diameter were fixed in 4 % (v/v) paraformaldehyde in phosphate buffer (pH 7.4), stained with propidium iodide in phosphate-buffered saline, immersed in 2 μM pyrene probe solution [
      • Valanciunaite J.
      • Kempf E.
      • Seki H.
      • et al.
      Polarity mapping of cells and embryos by improved fluorescent solvatochromic pyrene probe.
      ], and subjected to optical clearance using a transparency-enhancing technology (OCTET) [
      • Mizutani H.
      • Ono S.
      • Ushiku T.
      • et al.
      Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: a porcine model.
      ] for 48 h at room temperature. Images were acquired by two-photon excitation microscopy (TPM: Nikon A1RMP + and Spectra physics MaiTai DeepSee eHP) as described previously [
      • Murakami M.
      • Kawakami R.
      • Niko Y.
      • et al.
      High-quality fluorescence imaging of the human acrosyringium using a transparency: enhancing technique and an improved, fluorescent solvatochromic pyrene probe.
      ]. In briefly, Excitation laser was set at 960 nm and emission was divided by using dichroic mirrors at 495 nm and 560 nm. After TPM, all samples were processed for routine histopathology, embedded in paraffin blocks, cut into 3-μm-thick slices, stained with hematoxylin and eosin, and viewed under a microscope.
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