2.1.

Chemical Agents and Animal Preparation

The OCA is still based on the previous study,⁷ which is composed of two biocompatible agents, PEG-400 and Thiazone, and the volume ratio of PEG-400 and Thiazone is $9:1$.²⁹ The refractive index of the mixture solution is 1.47 (WAY-2S ABBE Digital Refractometer, Shanghai YiCe Apparatus & Equipment Co., China). Saline was also used as a control agent.

Animal care and experimental procedures were approved by the Experimental Animal Management Ordinance of Hubei Province, P. R. China. Male Sprague-Dawley (SD) rats ($n=50$, $105\pm 12\mathrm{g}$, 4 to 5 weeks old) were supplied by Wuhan University Center for Animal Experiment (Wuhan, China) and fed under specific pathogen free (SPF) level of feeding condition.

Rats were anesthetized with the mixture of 2% $\alpha$-chloralose and 10% urethane ($8\mathrm{mL}/\mathrm{kg}$) via intraperitoneal injection. The dorsal hair was shaved and the residual hair was removed with depilatory cream (sensitive hair removal cream, Veet), and then impurities of the region of interest were gently removed by tape stripping 2 to 3 times. After that, rats were randomly divided into two groups: the control group ($n=10$) and the experimental group ($n=40$). The experimental group received topical application of warm OCA (37°C), whereas the control group just received warm saline (37°C). The OCA or saline were removed from the skin 30 min later with lukewarm water (37°C). After experiments of the day, rats were back to the standard feeding environment.

In the following 28 days, rats were examined daily for any abnormal appearances of the skin and general reaction, which include awareness, motor activity, posture and muscle tone, etc. The body weight of the rats was recorded before the experiment and once every seven days after treatment of agents.

2.2.

LSCI for Imaging Dermal Blood Vessels

In this study, the cutaneous microcirculation was monitored by LSCI technique. Figure 1 shows the schematic of LSCI system. A 632.8-nm He-Ne laser (3 mW) was used to illuminate the interested area with a beam expanded through a collimating lens. A sequence of raw speckle images was then imaged through a stereo microscope (SZ61TR, Olympus, Japan) onto a charge coupled device (CCD) camera (Pixelfly USB, PCO Computer, Germany). The CCD exposure time used was 5 ms. Because the laser speckle temporal contrast analysis (LSTCA) method was reported to be highly effective for suppressing the influence of static speckle from the scattering medium,⁹ it was used here to obtain the two-dimensional (2-D) blood flow distribution maps of cutaneous microvessels.

Fig. 1

Schematic of the laser speckle contrast imaging system.

Forty rats were used for dermal blood flow imaging. After the prepared animal model was placed on the experimental platform, the initial raw speckle images of the interested skin area were obtained. Then, the OCA was topically applied on rat dorsal skin in vivo, and a sequence of raw speckle images were obtained at 2-min intervals. After 30 min, the OCA was gently removed and the saline solution was topically applied on the same area for temporary recovery. The raw speckle images were taken again. During the intervals, a digital camera (T200, Sony, Japan) was used to take the white-light photos. To reduce the side effects of OCA on rat skin, after the interested skin area was washed by saline solution, 0.5% hyaluronic acid (HA) and Vaseline were topically applied to keep skin moist and to prevent the contact of skin and bacterium.³⁰31.^–32 On the second and third day, the above experimental procedure was performed repeatedly.

2.3.

Histopathologic Examinations for Evaluating the Safety of OCA

Ten animals on the first day, the 14th day, and the 28th day after treatment of OCA for three days, were sacrificed, respectively. The skin of interest was cut and immersion-fixed in 4% neutral formaldehyde, and then dehydrated and embedded in paraffin. After cutting into 4-μm sections, the samples were then stained with hematoxylin and eosin (H&E), and Masson, respectively, and examined under a light microscope (IX71, Olympus, Japan).

On the 28th day after treatment of OCA and saline for three days, 10 rats from the experimental group and 10 from the control group were sacrificed, and various organs, such as liver, heart, spleen, kidney, brain, and lungs were removed and weighed. The organ coefficient was obtained by dividing the weight of the organ by body weight.

Both liver and kidney are important target organs of toxic effects of chemicals because they are primarily involved in metabolism and excretion of chemicals.³³ In this work, the liver and kidney tissue were also tested by histopathologic examination with H&E staining.

3.1.

OCA Induced Switchable Skin Window on Rat In Vivo

The OCA was repeatedly applied on the same area of rat skin in vivo in the following 3 days, and the typical results are shown in Fig. 2. Figure 2(a) shows the morphologic photos of the skin before, 30 min after application of OCA, and recovery with saline. Figure 2(b) shows the corresponding laser speckle temporal contrast images, which demonstrate the flow distribution information of the dermal blood vessels.

Fig. 2

Typical visual photos (a) and corresponding laser speckle temporal contrast images (b) of in vivo rat skin. The arrows 1 to 4 indicate the interested blood vessels.

It can be seen that on the first day topical application of OCA on rat skin in vivo makes the skin become transparent, which allows to image the dermal blood flow by the LSCI technique; whereas treatment with saline makes the skin recover to its initial state, which is consistent with the previous study.⁷ On the second or the third day, the initial state of rat skin is almost the same as the intact skin, without wrinkles and edema occurring in the skin. It is difficult to image blood flow because the skin is turbid. After topical application of OCA on the same place, the skin becomes transparent again, and even the small branches of dermal blood vessels can be clearly observed. The details of dermal blood-flow distribution information are available to be acquired by LSCI technique with high resolution. With treatment of saline solution, the skin recovers to the initial state, and the blood vessel structure and flow information are concealed by the high scattering of skin once again. Comparing the transparent skin in the following two days with that of the first day, we can find that the vessel structure and flow distribution of dermal blood vessels are quite consistent.

To further quantitatively evaluate whether repeated administration of OCA affects the dermal blood vessels, the diameter of some typical vessels (pointed out by arrows 1–4) in Fig. 2 are calculated, and the results are summarized as $\text{mean}\pm \text{standard}$ deviation in Table 1. In addition, a one-factor analysis of variance (ANOVA) was applied for statistical analyses, and the differences in vessel diameter between the following two days and the first day were inspected by least significant difference (LSD) test, respectively. It can be found that, after repeated application of OCA on rat skin, the diameters of dermal blood vessels measured in the following two days are essentially unchanged from that measured in the first day, and there are no significant changes in diameter between day 2, day 3, and day 1 ($P>0.05$). That is, the OCA has no obvious side effects on the dermal blood vessels. Thus, it seems to be a switchable window that topical administration of OCA or saline on rat skin in vivo can open or close the skin window. It should benefit from the excellent moisturizing and bacteriostatic efficacy of HA and Vaseline, which permits the blood vessel structure and flow distribution to be imaged repeatedly without surgical operation.

Table 1

Measurement of diameter of dermal blood vessels shown in Fig. 2.

3.2.

Short-Term and Long-Term Effects of OCA on the Microstructure of Skin

The above results demonstrate that a switchable skin window can be established with the optical clearing method, but the safety of OCA to the skin and body need to be evaluated. Figure 3 shows the results of histopathologic examinations of rat skin before and after treatment by OCA. The H&E stains of skin sections in the top row show the microstructure of skin, and the Masson stains of skin sections in the bottom row show the collagen alignment in dermis. Figure 3(a) and 3(e) shows the control skin sections; others in Fig. 3 show the skin sections on the first day, the 14th day, and 28th day after treatment of OCA for three times, and the arrows 1 to 3 in Fig. 3(a) point out the structure of the stratum corneum, epidermis, and dermis, respectively.

Fig. 3

H&E stains (top row) and Masson stains (bottom row) of tissue sections of rat skin. (a) and (e) Control skin sections; (b) and (f), (c) and (g), and (d) and (h) skin sections on the first day, the 14th day, and the 28th day after treatment of OCA for three times. The arrows 1 to 3 point out the stratum corneum, epidermis, and dermis, respectively.

It can be found that, for control skin, the structure of epidermis and dermis is integrated. On the third day, after treatment of OCA for 30 min, the stratum corneum is destroyed, and no inflammation cells are found in epidermis and dermis. Moreover, the epidermis becomes thin, whose thickness decreases from $59.36\pm 8.68\mu \text{m}$ to $21.06\pm 2.31\mu \text{m}$. However, on the 14th day after treatment by OCA for three days, the stratum corneum of rat skin recovers to normal. On the 28th day after treatment, the good condition remains, and there are no obvious differences in the microstructure of rat skin between the control skin and the OCA-treated skin.

Previous studies showed that the process of skin optical clearing might be accompanied by structural modification or dissociation of collagen.³⁴ Hence, Masson stains of skin sections after application of OCA were also conducted to see the changes in collagen alignment. The results show that, for control skin, the collagen is arranged densely. After application of OCA for three days, the arrangement of collagen is unchanged compared with the control skin. On the 14th day and the 28th day after treatment with OCA three times, the collagen alignment is also unchanged. That is, no histopathologic or microstructural changes in rat skin are induced by administration of OCA.

3.3.

Effects of OCA on the Body and Organs

Further, we performed toxicity experiments to demonstrate whether there is potential toxicity of OCA to the rat body and various organs.

To evaluate the influence of OCA on the body and target organs, the weights of body and various organs, i.e., liver, heart, spleen, kidney, brain, and lungs, etc., were obtained, and the organ coefficients were calculated. Data of body weight and organ coefficients were summarized as $\text{mean}\pm \text{standard}$ deviation in Table 2. A one-factor analysis of variance (ANOVA) was applied for statistical analyses, and the differences between the experimental group and control group were inspected by the Q method (student-Newman-Keul test). It can be found that the body weight has almost doubled in the four-week observation period, but there are no significant differences in body weight and organ coefficients between the control group and the experimental group ($P>0.05$).

Table 2

Measurement of body weight and organ coefficients of rat after treatment with OCA.

Furthermore, on the 28th day after treatment of OCA or saline, both the kidney and liver were histopathologically examined by H&E staining, respectively. Figure 4 shows the typical sections of kidney (top row) and liver (bottom row). The left column shows the control samples and the right column shows the experimental samples. It can be seen that after treatment by OCA, the cortical architecture of kidney tissue remains intact. Compared with the control section, neither inflammatory infiltrate nor abnormal histopathologic changes were observed in glomeruli pointed out by arrow 1 in Fig. 4(a) and 4(b). Compared with normal liver tissue, the OCA-treated hepatic tissue section has no pathological abnormalities. The hepatocytes pointed out by arrow 2 are arranged in organized plates with well-formed sinusoids pointed out by arrow 3 in Fig. 4(c) and 4(d) in between.

Fig. 4

H&E stains of kidney (top row) and liver tissue (bottom row). (a) and (c) Control tissue sections; (b) and (d) Tissue sections on the 28th day after treatment with OCA for three days. The arrows 1 to 3 point out the structure of glomeruli, hepatocytes and sinusoid, respectively.