Clinical & histological assessment
TAA, atrophy, white striae/no, pain
STERILE, ATROPHY, ATROPHIC
Patients were evaluated in 5 visits over the course of the 12-week study: at baseline (after 7 days from diagnostic biopsy), at the end of the 2nd, 4th, and 8th weeks of treatment, and after 4 weeks of treatment cessation (12th week).The clinical effectiveness was evaluated using the clinical scoring (CS), in which “0” represented no lesion/normal mucosa; “1” mild white striae/no erythematous area, “2”white striae with an atrophic area less than 1 cmPhotographs of the oral marker lesion for each patient and in every visit were taken using the same digital camera with the same settings (Panasonic Lumix DMC-FH1, resolution 4000 × 3000, 12 mega pixels, 5× optical zoom, with auto macro focus range of 5 cm, Panasonic. Co.). Digitalized images were taken for the analysis using (Adobe Photoshop CS3 Extended version 10.0. Adobe Systems Inc. USA). The total areas of atrophy (TAA) in each marker lesion were outlined and measured in pixels, then transformed into actual surface areas in mmPatients also ranked the severity of pain and burning sensation on a 100-mm visual analogue scale (VAS) [On two occasions, a punch biopsy using a sterile single-use biopsy punch (Kai industries co., ltd. Japan) for approximately 4 mm in width and 5 mm in depth was carried out at the first evaluation visit and at the same site after 8 weeks of treatment. The biopsy site within the marker lesion for each patient was selected so as to avoid areas completely denuded of epithelium and to include keratotic areas and part of normal looking mucosa.Specimens were fixed in 10% buffered formalin solution, dehydrated, and embedded in paraffin blocks for hematoxylin and eosin staining for initial diagnosis. Other sections were prepared on positively charged glass slides; in summary, immunohistochemical staining is performed as follows: The formalin-fixed, paraffin-embedded specimens in the other block are subsequently cut into 3-µm-thick sections using a tissue microtome, and mounted on aminosilane-coated positively charged glass slides. Sections were then deparaffinized and rehydrated with a graded ethanol series before being immunostained with a labelled streptavidin–biotin immunohistochemistry assay (Lab Vision Corporation, United Kingdom, Ultra-Vision Detection System Anti-Polyvalent, HRP/DAB (Ready-To-Use) TP-015-HD). Photomicrographs of tissue sections were captured at a magnification of 20× using a digital video camera (Canon EOS 650D, Japan) mounted on a light microscope (BX60, Olympus, Japan). For immune-stained sections in each positive section at least four microscopic fields showing the highest immunopositivity were selected (nuclear staining of any intensity was considered positive, and blue nuclear staining was considered negative). Photomicrographs were then transferred to the computer system for analysis for both the number and localization of Caspase-3 immuno-positive cells in the lining epithelium, and in the underlying connective tissue using image analysis software (Image J, 1.41a, NIH, USA). The area fraction of the positive cells, then the mean area fraction (MAF) representing the mean of percentage of immunopositive area to the total area of the four microscopic field, lymphocytic cell counts for selected fields were also calculated automatically.