Subclinical effects of botulinum toxin A and microwave thermolysis for axillary hyperhidrosis: A descriptive study with line‐field confocal optical coherence tomography and histology

Botulinum toxin A (BTX) and microwave thermolysis (MWT) are standard axillary hyperhidrosis treatments, but comparison of their subclinical effects is lacking. Line‐field confocal optical coherence tomography (LC‐OCT) is a promising non‐invasive imaging tool for visualizing tissue‐interactions. This study aimed to describe subclinical effects of BTX and MWT for axillary hyperhidrosis with LC‐OCT‐imaging compared to histology. This study derived from an intra‐individual, randomized, controlled trial, treating axillary hyperhidrosis with BTX versus MWT. Subclinical effects based on LC‐OCT images from baseline and 6‐month follow‐up (n = 8 patients) were evaluated and compared to corresponding histological samples. At baseline, LC‐OCT visualized eccrine pores at the skin surface and ducts in the upper dermis (500 μm), but not deeper‐lying sweat glands. Histology identified entire sweat glands. Six months post‐treatment, LC‐OCT revealed no detectable morphology changes in any BTX‐treated axillae (100%), while recognizing obstructed eccrine pores and atrophy of eccrine ducts in most MWT‐treated axillae (75%). Histology corroborated LC‐OCT findings, while also showing substantial changes to entire sweat glands. LC‐OCT enabled visualization of subclinical alterations of superficial eccrine ducts after MWT and unchanged morphology after BTX. LC‐OCT is a promising tool for non‐invasive assessment of treatment‐specific tissue‐interactions that can be complementary to histology.


| BACKG ROU N D
Primary axillary hyperhidrosis management petitions for both better treatment monitoring and outcome assessment.Botulinum toxin A (BTX) and microwave thermolysis (MWT) are two well-established treatments for axillary hyperhidrosis with very distinctive clinical profiles as a neurotoxin and an energy-based procedure, respectively. 1Clinically, BTX is known to have a temporary effect while MWT can initiate more extensive changes that may be permanent.3][4] However, the nature of histological sampling is invasive, partial and scarring, which limits its utility for continuous monitoring of delimited treatment areas such as the axillae.Furthermore, collection of histological samples is time-consuming and resource-heavy.
The prospect for real-time, in-vivo, and non-invasive assessment of treatment effects remains to be explored with the most recently developed imaging devices.
Line-field optical coherence tomography (LC-OCT) is a newer imaging technology that combines the optical principles of optical coherence tomography (OCT) and Reflectance Confocal Microscopy. 5 This allows for high-resolution images of the superficial skin on a cellular level down to the upper dermis.New studies are investigating the utility and versatility of LC-OCT in a variety of skin diseases, [6][7][8][9][10][11][12][13] but none yet in hyperhidrosis.Comparison of subclinical effects after different, available treatments, is relevant for precise treatment outcome assessment.LC-OCT may provide a new, in-vivo, non-invasive alternative for assessment of subclinical features and treatment-specific tissue-interactions.

| Aim -questions addressed
This pilot study aimed to (i) establish the feasibility and investigate the range of LC-OCT for visualization of the sweat gland unit, and (ii) explore the potential of LC-OCT for recognition of treatmentspecific tissue-interactions after axillary hyperhidrosis treatment with BTX versus MWT, compared to histology.The findings presented in this paper are from a substudy within the above-mentioned trial.The clinical trial has previously been reported on in detail, 14 and the substudy was assembled for additional subclinical outcomes with LC-OCT imaging and histological samples.

| Study design
Substudy participants were prospectively recruited on a volunteer basis, agreeing to undergo LC-OCT imaging and biopsies from each axillae at baseline and repeated at 6-months post-treatment.Separate informed consent forms were obtained for inclusion in the substudy.
Consents for the use of images, including photographs, were also secured.Study participants were included in the substudy before treatment randomization in order to reduce bias on outcome assessment.

| LC-OCT imaging
LC-OCT imaging of the mid-axillary region was performed by one operator (KJ) using the handheld deepLive™ probe (DAMAE Medical, Paris, France). 5 The LC-OCT is an optical non-invasive imaging technique using a broadband light-source centered at ~800 nm providing a penetration depth down to 500 μm.Three-dimensional (3D) grey scale images are obtained by stacking either vertically or horizontally oriented images, and real-time videos can be recorded.
To ensure reproducible images of the mid-axillary site over time, a transparent template was placed within the clinically pre-defined treatment areas (see Figure S1 for setup).The template consisted of three 3-mm sized cut-out holes.Each hole was separately LC-OCT imaged at baseline and 6-months, using a combination of LC-OCT video recording and 3D image-acquisition.Thereafter, all images were evaluated qualitatively by one physician with experience in LC-OCT (KJ).

| Histology
Histological samples were collected as the 'gold standard' for comparison with LC-OCT findings.In the mid-axillary area, beside the imaging areas, a 4 mm biopsy was obtained from each axilla at baseline.At 6-months follow-up, the baseline biopsy locations were identified, and in close proximity, avoiding any visible scar tissue, a follow-up biopsy in each axilla was collected.All histological samples were stained with Haematoxylin and Eosin (HE) in a standardized manner.Blinded to the randomization result, a dermatopathologist (NM) qualitatively assessed the paired samples at baseline and 6 months after BTX and MWT, respectively.

| Descriptive outcomes
The range of LC-OCT for recognizing sweat gland unit structures in the axillae was evaluated.First, by establishing the presence or absence of recognizable sweat gland structures at baseline in LC-OCT images.The histological samples were used as a reference point for confirmed presence of typical axillary sweat gland structures.
The treatment-specific tissue-interactions in the axillae were represented by observable morphological changes of the sweat gland unit.They were qualitatively described in both LC-OCTimages and histological samples, comparing baseline to 6-months after MWT-and BTX-treatment, respectively.

| RE SULTS
A total of eight patients, comprising 16 axillae, were included.LC-OCT images and biopsies were collected for all patients both at baseline and 6 months post-treatment with BTX in one axilla and MWT in the contralateral axilla, respectively.All LC-OCT images were feasible for descriptive assessment, while in a single patient, follow-up biopsies were not representative and thus could not be evaluated properly (see Table S1 for tabular overview of the described findings from LC-OCT and histology).

| Baseline
In all axillae, LC-OCT could identify the eccrine sweat ducts down to upper dermis (500 μm) as well as the pores on the skin surface in detail, both in the vertical and more pronounced in the horizontal (en-face) plane (see Figure 1A).The ducts presented with a characteristic simple, coil-like shape from the depth to the surface of the  Histology confirmed comparable conditions between sides before treatment, with well-defined sweat glands in all samples.Eccrine sweat glands appeared normal in density and size with well-defined lumen and epithelium.Apocrine sweat gland structures were also visualized with typical appearance, some with slightly dilatated lumen, but all with normal epithelium (see Figure 2A).
Correspondingly, BTX-treated samples did not show any histological changes compared to baseline (see Figure 2B).Histologically, some patients presented with slight dilatation of the apocrine sweat glands, but no different to their corresponding baseline.
LC-OCT could identify the residual superficial eccrine gland structures (see Figure 1C) after MWT-treatment.In the majority of MWT-treated axillae (n = 6, 75%), obstructed pores and atrophy of eccrine ducts were visualized, while in the remaining axillae (n = 2, 25%) features were poorly defined, limiting the tissue-interaction assessment.Generally, the eccrine duct epithelium appeared atrophic in the MWT-treated axillae, but fibrosis could not be identified with certainty.
Histologically, after MWT, there was visible reduction in sweat gland density and there was atrophy of eccrine sweat glands with shrinkage in size and shrinkage of the basement membrane.
Surrounding fibrosis with loss of elastin fibres was also identified (see Figure 2C).Apocrine glands could not be detected in all follow-up samples.Recognizable apocrine glands were identified in a single MWT-sample, but they presented atypical with massive dilatation and flattening of the epithelium.

| CON CLUS I ON S & PER S PEC TIVE S
To our knowledge, LC-OCT-imaging in axillary hyperhidrosis as well as comparison of treatment-specific tissue-interactions following MWT and BTX specifically, has not previously been described.
LC-OCT can identify the superficial ducts and pores from eccrine sweat glands in the axillae down to the upper dermis at 500 μm.
The images' resolution also allows for visualization of the duct epithelium.Both in LC-OCT-images and the histological samples, no visible changes after BTX were detected, while subclinical changes, such as atrophy of the sweat gland structures, after MWT could be described.In the corresponding clinical trial, a significant sweat and odor reduction after both treatments was reported at 6-months post-treatment, 14 but on the long-term (12 months) majority of patients preferred the overall effect of MWT due to the potential of sustained effect.From a clinical perspective, BTX is considered a temporary treatment, but the effect of consecutive and/or repeated injections remains unclear.In this study we did not find any indication that BTX led to tissue-interactions causing structural changes in the sweat gland unit.Half of the study patients were naïve to previous BTX-treatments, while the other half had received BTXtreatments prior to inclusion, but no difference in patient outcomes was observed.Non-invasive assessment of long-term subclinical effects from BTX and MWT could be an interesting topic for future studies.
In comparison to the limited resolution of traditional Optical Coherence Tomography (OCT), 15 LC-OCT provides a detailed visualization of eccrine gland structures.However, LC-OCT is simultaneously limited by its depth range of 500 μm which only allows visualization of the superficial part of the eccrine sweat gland unit.Additionally, apocrine sweat gland structures could not be surely distinguished from the hair follicle and shaft, even though the infundibulum corresponds to the superficial part of the hair follicle.Tissue-interactions after MWT was detected with LC-OCT by a trained physician in the majority of cases.However, in some images, the structures were insufficiently defined for certain identification of subclinical changes.This was deemed due to difference in image quality as the particular cases had clinical outcomes comparable to the cases with well-defined changes.
Future application of machine learning on larger data sets could potentially be able to distinguish poorly defined features in order to achieve higher rate of outcome assessment.Additionally, quantitative data analysis may also be facilitated in the future with the application of relevant pattern recognition on the LC-OCT videofunction.Another important consideration for future use of realtime, non-invasive imaging, is the depletion of sampling errors and non-representative biopsies.Finally, if LC-OCT could be considered a robust surrogate marker, a potential reduction in the need for invasive biopsies would improve the conditions and feasibility for future studies altogether.
In conclusion, the corresponding tissue-interactions identified in this study support the clinical profiles of temporary, non-structural changes from BTX and irreversible tissue changes from MWT. Due to LC-OCTs non-invasive nature, it has the potential to become a valuable tool for in-vivo assessment of treatment effects by description of treatment-specific tissue-interactions.

A
prospective, randomized, within-patient, controlled trial investigating BTX versus MWT for axillary hyperhidrosis was carried out between 2021 and 2023 at the Department of Dermatology, Copenhagen University Hospital, Denmark, in accordance with the Helsinki Declaration.The study was approved and registered by all the required authorities; The Danish Medicines Agency, EudraCT: 2021-000877-10, The Regional Health Research Ethics Committee in Copenhagen, H-21013548, The Danish Data Protection Agency, P-2021-436, and on Clini calTr ials.gov NCT05057117, ID: MWT-BTXA.
skin.Eccrine ducts opened to the skin surface as tubular pores.The duct epithelium could be identified.The deeper-lying eccrine glands were not visible with this technique, and apocrine sweat gland structures could not conclusively be distinguished from the hair follicle and shaft due to lack of resolution.F I G U R E 1 Sweat ducts and pores visualized with LC-OCT at (A) baseline and 6-months after (B) BTX-treatment and (C) MWT-treatment.(A) Well-defined eccrine sweat gland unit visualized en-face in the epidermis and superficial dermis; left panel: eccrine pore opening on the skin surface visible at 30 μm depth (white arrow); right panel: deeper duct epithelium well-defined (cyan arrow) with surrounding collagen and central coil-shaped eccrine duct visible at 100 μm depth (yellow arrow).(B) Well-defined eccrine duct morphology of the BTX-treated axillae visualized en-face in the epidermis and superficial dermis: left panel, layered eccrine pore opening on the skin surface visible at 20 μm depth (white arrow); right panel, layered duct epithelium well-defined (cyan arrow) with central coil-shaped eccrine duct visible at 80 μm depth (yellow arrow).(C) Subclinical effects after MWT-treatment visualized en-face in the epidermis and superficial dermis: left panel, eccrine pore opening on the skin surface obstructed with skin debris visible at 20 μm depth (white arrow); right panel, deeper duct epithelia not well-defined and atrophic (cyan arrow) with remnant central coil-shaped eccrine duct visible at 110 μm depth (yellow arrow).BTX, botulinum toxin A; LC-OCT, line-field optical coherence tomography; MWT, microwave thermolysis.

2
Sweat ducts and glands visualized with histology at (A) baseline and 6-months after (B) BTX-treatment and (C) MWTtreatment.(A) Sweat gland structures visualized in the lower dermis at baseline; left panel: section overview; right panel: normal eccrine sweat ducts and glands (yellow arrows).Apocrine sweat glands with normal epithelium, some with slight dilatation (green arrows).(B) Sweat gland structures visualized in the lower dermis after BTX-treatment; left panel: section overview; right panel: normal eccrine sweat ducts and glands (yellow arrows) with no atrophy or surrounding fibrosis.Apocrine sweat glands with normal epithelium, some with slight dilatation (green arrows).(C) Sweat gland structures visualized in the lower dermis after MWT-treatment; left panel: section overview; right panel: atrophic eccrine sweat ducts and glands (yellow arrows) with surrounding fibrosis (red arrows).No recognizable apocrine sweat glands.BTX, botulinum toxin A; MWT, microwave thermolysis.