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

Abstract

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 declare no conflicts of interest.