ORIGINAL STUDY Abnormal Morphology of Blood Vessels in Erythematous Skin From Atopic Dermatitis Patients Moe Tsutsumi, PhD,*† Maki Fukuda, BS,† Junichi Kumamoto, BS,*‡ Makiko Goto, PhD,*† Sumiko Denda, PhD,*† Kenshi Yamasaki, MD, PhD,§ Setsuya Aiba, MD, PhD,§ Masaharu Nagayama, PhD,*‡ and Mitsuhiro Denda, PhD*† Abstract: Previous studies suggest that altered peripheral blood circulation might be associated with erythema or inflammation in atopic dermatitis (AD) patients. However, the overall structure of blood vessels and capillaries in AD skin is poorly understood because most studies have involved light-microscopic observation of thin skin sections. In the present study, we compared the 3-dimensional structures of peripheral blood vessels of healthy subjects and AD patients in detail by means of 2-photon microscopy. In skin from healthy subjects, superficial vascular plexus and capillaries originating from flexous blood vessels were observed. However, skin from AD patients contained thickened, flexuous blood vessels, which might be associated with increased blood flow, in both erythematous and nonlesional areas. However, patients with lichenification did not display these morphological changes. Bifur- cation of vessels was not observed in either erythematous or lichenification lesions. These results might be helpful for developing new clinical strategies to treat erythema in AD patients. Key Words: two-photon microscopy, peripheral blood vessel, vas- cular plexus (Am J Dermatopathol 2016;38:363–364) INTRODUCTION In the dermis of normal subjects, the vascular system is organized into a deep and superficial horizontal plexus from which capillaries originate. 1 In acute inflammation, blood vas- cular endothelial cells are activated by multiple inflammatory mediators [such as vascular endothelial growth factor (VEGF)], leading to typical signs of inflammation as a result of both increased blood flow because of vessel dilation and edema formation because of increased vessel permeability. For example, the number and size of blood vessels are increased in inflamed skin of K14-VEGF-A transgenic mice compared with uninflamed skin. 1 Accordingly, previous re- ports suggest that altered peripheral blood circulation might be associated with erythema in atopic dermatitis (AD) pa- tients. For example, the amount of VEGF in lesional scales in AD was 25 times higher than that in normal stratum cor- neum. 2 Steinhoff et al 3 suggested that the vascular system, including endothelial cells and smooth muscle cells, is inti- mately involved in the clinical symptoms of AD, including erythema, edema, leukocyte recruitment, and white dermog- raphism, and they proposed that endothelial cells play a cru- cial role in the pathophysiology of AD. These reports led us to hypothesize that the 3-dimensional structure of blood vessels and capillaries is altered in the erythematous skin of AD patients. To examine this question, light-microscopic observation of thin sections, as usually used, is inadequate because it cannot differentiate between increased vessel branching and increased total length of vessels. Therefore, we used 2-photon microscopy, which is a fluorescence imaging technique that allows imaging of tissue to a considerable depth to compare the 3-dimensional morphologies of blood vessels in the upper layer of dermis of healthy subjects and AD patients. METHODS Sample Collection Skin samples were obtained from 10 Japanese healthy subjects and 9 AD patients with lichenification (20–37 years; mean age of 24 years; 13 men and 6 women). The study protocol was fully discussed with each patient and informed consent was obtained. The study was conducted in accor- dance with the guidelines of the National Institutes of Health and Declaration of Helsinki protocols and was approved by the ethics committees of Shiseido Research Center and Tohoku University Hospital. Tape stripping was performed 10–30 times under local anesthesia with lidocaine, and then a 3-mm punch biopsy was obtained from the back skin. In the case of AD patients, samples were taken from regions of erythema or lichenification and noninvolved regions. The specimens were immediately immersed in 4% paraformalde- hyde overnight at 48C. Immunohistochemical Study After the 4% paraformaldehyde treatment, samples were kept in 30% sucrose until they sank to the bottom. Then, one half of each sample was embedded in optimal cutting temper- ature compound (TissueTek; Sakura Finetek, Torrance, CA) and From the *Japan Science and Technology Agency, CREST, Kawaguchi, Japan; †Shiseido Research Center, Yokohama, Japan; ‡Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan; and §Depart- ment of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan. The authors declare no conflicts of interest. Reprints: Mitsuhiro Denda, PhD, Shiseido Research Center, 2-2-1, Hayabuchi, Tsuzukii-ku, Yokohama 224-8558, Japan (e-mail: mitsuhiro.denda@to.shiseido.co.jp). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Am J Dermatopathol  Volume 38, Number 5, May 2016 www.amjdermatopathology.com | 363 Copyright © 201 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. 5