In Vivo Non-Invasive Observation of Dynamic Changes in Hair Shaft, Melanin, and Collagen During the Mice Hair Follicle Cycle

Summary

This study presents a non-invasive, in vivo method using two-photon and second-harmonic generation microscopy to longitudinally monitor hair shaft morphology, melanin dynamics, and dermal collagen density throughout the entire hair follicle cycle in mice, providing a robust methodological framework for hair-loss therapeutic research.

Abstract

Traditional evaluations of the hair follicle cycle rely on invasive histological methods, limiting continuous longitudinal observation. It was hypothesized that non-invasive optical imaging, particularly two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG), can accurately track and quantify in vivo dynamic changes in hair shafts, melanin, and collagen across multiple hair follicle cycles. The hair shafts of C57BL/6 mice were monitored in vivo during the anagen, catagen, and telogen phases using dermoscopy, photography, and electron microscopy. Specifically, TPEF under 800 nm excitation and imaging were used to quantify melanin dynamics, while SHG under 950 nm excitation was used to observe interfollicular collagen changes. To ensure accuracy, the in vivo optical measurements were cross-validated: melanin dynamics were confirmed via skin photography for overall tissue appearance and hematoxylin and eosin (HE) staining for hair bulb expression, while collagen changes were validated by assessing fiber density and orientation using a liquid crystal polarizing imaging system on Sirius Red-stained sections. The findings observations demonstrated that during the anagen phase, hair shaft diameter and melanin production progressively increased, accompanied by a significant reduction in interfollicular collagen density. During the catagen and telogen phases, melanin levels gradually decreased, and collagen density gradually recovered to baseline levels. This project establishes a validated, non-invasive in vivo methodology for evaluating the comprehensive dynamics of hair follicles, providing a robust experimental framework for the preclinical evaluation of active agents for hair loss treatments.

Introduction

The hair follicles of C57BL/6 mice exhibit synchronicity and regularity, offering experimental convenience and making them a common animal model for studying skin diseases^1,2. The hair follicles enter the second telogen phase in the seventh week after the birth of the C57BL/6 mouse³. In the absence of artificial intervention, this telogen phase will be maintained until the twelfth week after birth⁴. This extended period of telogen is a common time point used in research to synchronize the hair follicle growth cycle⁵. After artificial depil....

Protocol

All methods that involve the use of vertebrate subjects were performed in compliance with institutional guidelines. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Experimental Research Center of China Academy of Chinese Medical Sciences (Protocol Code: ERCCACMS11-2210-03). All the materials used in this study are listed in the Table of Materials.

Animal preparation and macroscopic observation
Male C57BL/6 mice, aged 6–7 weeks and weighing 20 ± 2 g, were obtained from a commercial supplier. The mice were divided into a histological ....

Results

Mouse hair observation
Photographic observations of murine hair growth are presented in Figure 1A. Hair emergence through the skin surface commenced on day 10, with continued development maintained between day 12 and day 16 (p < 0.05). Hair coverage subsequently plateaued from day 18 to day 26 (p > 0.05) (Figure 1B). Furthermore, dermoscopy facilitated the non-invasive in vivo observation of hair growth dynam.......

Discussion

The C57BL/6 mouse model was selected because its second telogen phase stably lasts from week 7 to week 12. Since the 26-day observation window falls entirely within this static phase, the profound dynamic changes observed are explicitly driven by the depilation-induced cycle progression, entirely excluding natural age-related fluctuations. When depilation is performed during the telogen phase, the hair follicles immediately enter the anagen phase. To the best of the author’s knowledge, this study presents a novel n.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 mice,	Beijing Vital River Laboratory Animal Technology	SCXK (Jing) 2021-0006	Used for animal models
Digital DSLR Camera	Canon	EOS 80D	Used to take pictures of the back of mice
H&E Staining Kit	LtdSolarbio Science & Technology Co.	G1120	Used for staining tissue sections
Inverted Microscope	Olympus	BX51	Used for taking photos
lsoflurane	RWD Life Science Co.	R510-22-16	Used for anesthetizing animals
Mai Tai DeepSee	SpectraPhysics	Mai Tai HP	Used for For TPEF and SHG in vivo imaging
Medical Electronic Dermatoscope	China Chuanghong Medical Technology Co.	CH-DSIS-2000	Used for in vivo imaging of skin color
Polarized Light Microscope	Olympus	BX51-P	Used for imaging Sirius Red staining
Scanning electron microscope (SEM)	Hitachi High-Tech Corporation	SU8010	For hair shaft ultrastructure observation
Sirius Red staining kit	Solarbio	G1472	For collagen fiber staining
Small Animal Anesthesia Machine	RWD Life Science Co.	R500	Used for anesthetizing animals