2.1.

Animal Model and Experimental Protocol

This work was approved by Huazhong University of Science and Technology Institutional Review Board for Animal Study. Female C57BL/6 mice ($n=30$), 3 to 3.5 weeks old, with the first postnatal telogen hair follicles,⁶ were obtained from Experimental Animal Center of Wuhan University (Wuhan, China) and fed under standard rodent feeding conditions. After 3 days of environmental adaptation, animals were intraperitoneally anesthetized with a mixture of chloral hydrate ($0.02\mathrm{g}/\mathrm{ml}$) mixed with ethylurethanm ($0.1\mathrm{g}/\mathrm{ml}$). To avoid possible effects of depilatory cream on the hair growth or cycle, the eudipleural areas of dorsal hair were shaved off by an electric shaver and then cleared carefully with a razor blade. In the next 23 days, the mice were photographed and the reflectance spectra of the back skin were measured every day.

2.2.

Histopathological Examination

To canonically discriminate the stage of hair follicle growth, histological examination was performed. Skin biopsies of mice dorsal skin were cut at different durations (3 to 7 weeks old). Three mice were sacrificed at each time point and the dorsal skin samples were detached and then fixed with 4% neutral formaldehyde and dehydrated with graded alcohol. After paraffin embedding, the samples were sliced with a thickness of 4 to $5\mu \mathrm{m}$ and then stained with hematoxylin and eosin (HE). The slice was imaged by a microscopy (Nikon Ni-E, Nikon, Japan) equipped with a color digital industrial camera (DS-Fi2, Nikon, Japan).

2.3.

Visible-Near-Infrared Fiber Spectrometer

To noninvasively obtain the change in mice skin with hair cycling, a visible-near-infrared (VIS-NIR) fiber spectrometer (USB-4000, Ocean Optics, Florida) was used to measure the diffuse reflectance spectra of in vivo mouse skin.¹² The system is composed of a light source (HL-2000), a reflection probe (R400-7), and a spectrometer as well as computer software. Before the experiment, the system was standardized with a reflectance standard film made up of barium sulfate. During this experiment, the probe was kept vertical and soft touched to the in vivo dorsal skin, then the data were saved. For each mouse, the eudipleural areas of dorsal skin were measured, respectively; for each area, the reflectance spectrum was measured three times and then averaged. Further quantification of reflectance was analyzed at a wavelength of 480 nm because of the most obvious and disciplinary changes in reflectance as well as the relatively low absorption of whole blood.¹⁷

2.4.

Pigmentation Evaluation of Mice Skin

Hemoglobin and melanin are major pigmentations in the skin. During hair follicle growth cycling, the skin color is changed from pink to gray, and then to black, which reflects different melanin contents in the skin. However, the absorption of hemoglobin will decrease sharply in the wavelength range of dark red or NIR, which is much less than that of melanin. In this work, the melanin value (dimensionless) was calculated from reflectance at 660 and 880 nm as follows:^18,19

2.5.

Quantitative Analysis

In order to quantitatively investigate the changes in reflectance and pigmentation of mouse dorsal skin with hair follicle cycling, the relative changes in reflectance ($\mathrm{\Delta }{R}_{\mathrm{rel}}$) at 480 nm and the melanin value ($\mathrm{\Delta }{M}_{\mathrm{v}\text{-}\mathrm{rel}}$) were obtained based on Eqs. (2) and (3), respectively. Here, $R_0$ and $M_{\mathrm{v}0}$ are the initial values on the first day of the experiment (D1), and $R_{\mathrm{t}}$ and $M_{\mathrm{vt}}$ are the values at different days during a hair follicle cycle.

3.1.

Macroscopical Changes in Mouse Dorsal Skin With Hair Follicle Cycling

Figure 1 show typical macroscopical changes in mouse dorsal skin during a hair follicle cycle. The dorsal skin is pink on the first day of the experiment (D1), which is at the beginning of the anagen stage. In the following several days, the color of the skin gradually changes from pink-to-gray. On the fifth day, the dorsal skin of the mouse seems to be gray. The skin color continues to darken as the anagen stage progresses, i.e., the dorsal skin shows dusty blue from the seventh to eighth day and dark blue from 11th to 15th day. Afterward, the skin color brightens to light blue (D18), gray-to-pink (D21), and finally back to pink (D23). Such cycling changes last for 23 days and roughly reflect the three stages of hair follicle growth, including anagen, catagen, and telogen. It can also be found that the skin color shows a synchronous change for both the tested area and the surrounding eudipleural areas.

Fig. 1

Macroscopical changes in mouse dorsal skin with hair follicle cycling. D means Day, D1 means the first day of experiment, which also corresponds to the starting date after hair follicles has transmitted to anagen stage, Dn means the day of the experiment. The speculated stages of hair follicle are generally shown as anagen, catagen, and telogen.

3.2.

Morphological Changes During a Hair Follicle Cycle

Figure 2 shows the skin tissue histological examination with HE staining. Since the morphological feature can precisely classify the stage of the hair follicle, the substages of the hair follicle cycle are indicated. On the first day of the experiment, the hair follicle is very short and is only located in the dermis. The faint expansion of dermal fibroblasts suggests that the hair follicle is at the anagen I. On the fifth day, the keratinocyte strands between the dermal papilla and club hair thicken with an obvious hair germ (Fig. 2, solid arrow head), which means that the hair follicle enters into the anagen II. On the eighth day, the hair follicles extend to the subcutis with an inflated hair bulb (Fig. 2, hollow arrow head). On the 11th day, the hair follicles reach to the bottom of the thickened adipose tissue with distinct dermal papilla, an outer root sheath (ORS) (Fig. 2, solid triangular arrow) and an inner root sheath (IRS) (Fig. 2, hollow triangular arrow), which means that hair follicles are from the anagen III to anagen VI. From the 15th to 18th day, the hair follicles reside in the deep subcutis, but the hair bulb gradually shrinks due to apoptosis of the matrix cells (Fig. 2, hollow arrow), which reveals the onset of the catagen stage. Afterward, further cell apoptosis leads to the absence of a multilayered structure (Fig. 2, double hollow arrows), and then enters into the telogen stage of the hair follicle.

Fig. 2

Development of the morphology of a hair follicle at different days and stages with hair follicle cycling. The solid arrow head indicates proliferating keratinocytes; the hollow arrow head indicates an inflated hair bulb; the solid and hollow triangular arrows indicate ORS and IRS, respectively; the hollow arrow reveals apoptosis of the hair matrix; the double hollow arrow reveals apoptosis of the cycling part of the hair follicle. Results were examined by HE staining. Scale bar: $100\mu \mathrm{m}$.

3.3.

Typical Changes in Reflectance Spectrum of Mouse Skin During a Hair Follicle Cycle

Typical reflectance spectra of mouse skin were measured within a hair follicle cycle [Fig. 3(a)]. On the first day, the reflectance of skin is very high; it begins to decrease sharply in 5 days. The reflectance spectra are at a low level from the 11th to 13th days. From the 15th day of the experiment, the reflectance begins to increase and reaches the initial level on the 23rd day. For higher reflectance spectra, obvious absorption peaks in the range of 500 to 600 nm can be found; whereas the absorption peaks almost disappear when the reflectances are at a lower level.

Fig. 3

Typical diffuse reflectance spectra (a) and its relative change ($\lambda =480\mathrm{nm}$) (b) of mouse skin with hair follicle cycling. Text arrows indicate different days (abbreviated as D) during a hair follicle cycle. Solid lines and lines with square indicate the decreased and increased trend of reflectance spectrum from the 1st to 13th and 15th to 23rd day, respectively. Data are shown as $\text{mean}\pm \mathrm{SD}$ ($n\ge 9$).

Further, relative changes in skin reflectance (480 nm) on different days compared with the first day were calculated. Since three mice were sacrificed for histological examination after every batch of measurements, only nine mice were left on the 23rd day after seven batches of measurement. Figure 3(b) shows the mean value and deviation from 9 to 30 mice. The results show that the relative change in reflectance is not obvious for the first few days; and then a decrease of 13% to 83% is shown from the 5th to 11th day. In the next a few days, the reflectance remains in a constant state. From the 15th day on, the reflectance begins to increase sharply and on the 23rd day is almost same as that on the first day.

3.4.

Skin Melanin Contents at Substages of Hair Follicle Cycle

Based on the reflectance spectra, the melanin values in skin on different days of the experiment were extracted using Eq. (1). Figure 4(a) shows that the melanin contents have a relatively low level during the first five days of the experiment, then increase rapidly and reach a relatively high level on the 11th day. Over the next several days, the melanin values reduce sharply and return to the initial value on the 23rd day.

Fig. 4

The skin melanin value calculated from reflectance spectroscopy during a hair follicle cycle (a). Relative change in melanin content on different days and corresponding substages of the hair follicle cycle (b). Data are shown as $\text{mean}\pm \mathrm{SD}$ ($n\ge 9$). Stages of hair follicles are subdivided into anagen I, II, III–IV, and VI, catagen, and telogen.

Further, the changes in melanin content relative to the first day of the experiment were calculated. After being compared with the results from the histological examination, the relative changes in melanin content at substages of the hair follicle cycle are indicated in Fig. 4(b). The melanin content in skin has very little change at the anage-І of the hair follicle cycle. On the fifth day, there is a 6% increase in the melanin value, which means that the hair follicle growth enters into the anagen II. Then the hair follicle growth quickly changes from the anagen III to IV on the seventh to ninth days. On the 11th day, the hair follicles enter into a later stage of growth (anagen VI) and the melanin value has an increase of $>60\%$. Once the hair follicles enter into the catagen stage (on the 15th to 21st days), the melanin value decreases rapidly, and goes down to the minimum value when hair follicles fall into the telogen stage (on the 23rd day).