New Characteristics of Eccrine Sweat Glands in Acquired Idiopathic Generalised Anhidrosis as Determined via Three‐Dimensional Fluorescence Imaging of Cleared Skin Tissue

Acquired idiopathic generalised anhidrosis (AIGA) is an acquired condition characterised by a noticeable decrease in sweating without an obvious cause [1]. The diagnostic guidelines for AIGA in Japan state that examination of a skin biopsy by optical microscopy and electron microscopy may demonstrate lymphocytic infiltration around the sweat glands and swelling of the secretory cells of the sweat glands; however, microscopy does not reveal marked morphological defects of sweat glands [1]. We treated a 47-year-old man with AIGA who presented with a 6-year history of reduced sweating involving the trunk and limbs. AIGA was diagnosed using the established criteria [1]. Blood tests, computed tomography, sialometry and Schirmer's test were performed. Differential diagnosis excluded neuropathy, exocrine gland dysfunction, and Sjögren's syndrome. A starch–iodine test confirmed anhidrosis affecting more than 95% of the trunk and limbs (Figure S1). Despite undergoing three courses of methylprednisolone pulse therapy during the past year, the patient's anhidrosis exhibited no improvement. We took 2-mm-diameter punch biopsy from an anhidrotic region of the lumbar back skin. Haematoxylin and eosin (H&E) staining revealed a lack of eccrine secretory glands. To investigate a potential decrease in sweat gland number due to atrophic changes, we took a 6-mm-diameter punch biopsy. The results were consistent with the initial examination, showing no eccrine secretory glands (Figure 1A). By contrast, tissue samples from patients without AIGA showed multiple glands within the same 6 mm range. Therefore, these results seem to imply that patients with AIGA may lose eccrine sweat gland structures. However, conventional H&E staining provides only two-dimensional images and has limitations in evaluating the total number and density of ducts and eccrine secretory glands because of their complex three-dimensional architecture. Therefore, we used a new three-dimensional fluorescence deep-imaging technique to compare the skin of patients with and without AIGA [2]. Briefly, we used HistoBright (Funakoshi, Tokyo, Japan), which is an improved solvatochromic fluorescent dye based on LipiORDER (Data S1) [3]. Sections of skin tissue were fixed with 4% paraformaldehyde in phosphate-buffered saline and stored frozen. Next, samples were stained with Hoechst and optically cleared using the LUCID method to enhance transparency and fluorescent dye penetration [4]. Finally, three-dimensional fluorescence imaging was performed using two-photon microscopy (TPM; AXE R MP; Nikon, Tokyo, Japan). The Ehime University Graduate School of Medicine Ethics Committee approved the study protocol, and the study was performed in accordance with the Declaration of Helsinki. Before evaluating the patient's skin, we imaged a 2-mm punch-biopsy lumbar back skin specimen from a patient without AIGA (Figure 1B–D). We confirmed the morphology and the numbers of acrosyringia (Figure 1C), dermal sweat ducts (Figure 1D) and eccrine secretory glands (Figure 1B). Notably, the TPM examination provided three-dimensional images of the eccrine sweat glands and enabled direct observation of the dermal sweat ducts extending toward the eccrine secretory coils (Video S1). TPM of the 6-mm punch-biopsy specimen from the patient with AIGA showed no acrosyringia (Figure 1C) or dermal sweat ducts (Figure 1D). The field of view covered 6 mm (Video S2), and there were no glands in the dermis or adipose tissue (Figure 1B). Thus, despite extensive observations, we did not find any structures related to sweat secretion. This is the first study to report the possibility of the disappearance of eccrine sweat glands in patients with AIGA based on an investigation using a three-dimensional, deep-imaging technique. Recent studies have reported the atrophy of sweat ducts, including eccrine secretory glands in AIGA patients [5, 6]. However, those studies used two-dimensional evaluation, which limited their assessment of the morphology, number, and density of three-dimensional structures. As mentioned above, HistoBright enables clear three-dimensional deep imaging of human eccrine sweat glands and acrosyringia. This method demonstrated the visualisation of the 3D morphology of the continuity between dermal sweat ducts and eccrine sweat glands without using immunofluorescence techniques. Our results revealed acrosyringia, dermal sweat ducts, and eccrine secretory glands in the skin of the patient without AIGA but none were found in the patient with AIGA. The gland density in the normal lumbar region has been reported to be 132 glands per cm2 [7]. Therefore, the inability to observe eccrine sweat glands within a 6-mm punch range is abnormal. Most importantly, this three-dimensional method of observation revealed either drastic decrease or disappearance of sweat glands, which was not possible using two dimensional imaging. Moreover, comprehensive evaluation of many patients with AIGA using this method may help to elucidate the causes of morphological abnormalities, potentially leading to a reassessment of treatment approaches. S.Y., R.K., and M.M. performed the research. Y.N. contributed essential reagents. S.Y., R.K., Y.F. and M.M. analysed the data. S.Y., R.K., Y.N. and M.M. wrote the paper. All authors read and approved the final manuscript. The authors wish to acknowledge Prof. Takeshi Imamura, Ehime University Graduate School of Medicine, for his kind advice about the interpretation of results. This work was supported by JSPS KAKENHI Grant Number JP22K06960. And, this work was supported by JST A-STEP Grant Number JPMJTR234G, Japan. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/jTg6aF. The authors declare no conflicts of interest. The authors have nothing to report. Figure S1. Evaluation of hidrotic areas using the starch–iodine test. Our patient exercised for 15 min. Although sweating was observed in the axillary regions, more than 95% of the surface area of our patient's body was anhidrotic. We took clinical photographs using an EOS Kiss X10 camera (Canon, Tokyo, Japan) with an EF50-mm F2.5 compact macro lens (Canon). The camera settings were adjusted to a shutter speed of 1/60 s and an aperture of f/25. Data S1. Details of methods. Video S1. Three-dimensional observation of sweat ducts and eccrine secretory coils of a patient without acquired idiopathic generalised anhidrosis (AIGA). Video S2. Three-dimensional observation revealing a lack of acrosyringia and dermal sweat ducts in a patient with acquired idiopathic generalised anhidrosis (AIGA). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.