2.1.

Preparation of Skin Samples

Skin specimens were obtained from the porcine abdomen in a local slaughterhouse within $1\text{-}\mathrm{h}$ postmortem. All samples were measured within $8\mathrm{h}$ and stored at $4°\mathrm{C}$ . The stratum corneum and the subcutaneous fat of the whole skin specimens were cleaned before the samples were dissected for measurements. The samples were cut into $50\times 50\text{-}\mathrm{mm}$ sections, and clamped tightly between two apertures of horizontal diffusion chambers. The diffusion chambers were made of two colorimetric wares. Each chamber was $50\times 50\times 5\mathrm{mm}$ , with an aperture of $30\mathrm{mm}$ in diam on one side, as shown in Fig. 1 . One chamber near the epidermis was filled with OCAs, and the other one was filled with isotonic saline. Both sides of the skin were able to react to the solution through the apertures. Because the osmotic pressure of isotonic saline was similar to in vivo physiological conditions, the diffusion chambers were able to simulate the physiological environment of skin in vivo.

Fig. 1

Experimental setup (sample box shows the diffusion chamber).

In this work, all samples $(n=50)$ were divided into seven groups, which included six experimental groups and a control group. The thickness of sample was measured by using a micrometer. The native thickness was about $1.61\pm 0.11\mathrm{mm}$ .

2.2.

Chemical Agents

The hydroxy-terminated agents have high OCP,⁹ small substances $(<500\mathrm{Da})$ easily penetrate the skin,²⁵ and the refractive indices of mammalian tissues are about 1.40,²⁷ so, in this study, we chose six alcohols with high refractive indices as OCAs, i.e., 1-butanol, 1, 4-butanediol, 1, 3-propanediol, PEG200, PEG400, and glycerol (Qiangsheng Chinese Chemicals, Limited, China). Their molecular weights range from $74\text{to}400\mathrm{Da}$ . At room temperature $(\sim 25°\mathrm{C})$ , the refractive index was measured with a refractometer (Digital Abbe Refractometer, WAY-2S, Shanghai, China). Table 1 shows refractive indices, molecular weights, and numbers of a hydroxyl group of the OCAs used in this study.

Table 1

Characteristics of the different alcohols and saline investigated in this study.

2.3.

Experimental System

An integrating sphere system was applied to monitor the transmission intensity of skin samples under the treatment of OCAs (see Fig. 1). A $1\text{-}\mathrm{mm}$ -diam beam He–Ne laser ( $\lambda =632.8\mathrm{nm}$ , $5\mathrm{mW}$ , Melles Griot, Carlsbad, California) was chopped mechanically at $1\text{-}\mathrm{kHz}$ (model SR 540), and then split into two beams by a beamsplitter. The reflected beam was a small fraction (20%) of the laser beam, which irradiated to a reference sphere of $70\mathrm{mm}$ in diam. The rest (80%) of the laser beam irradiated to the sample mounted in front of the port $(\varphi =25\mathrm{mm})$ of a big integrating sphere of $210\mathrm{mm}$ diam. The intensity of the transmitted light from the sample, including collimated and diffuse components of transmitted radiation, was collected by the integrating sphere. The signals from the Si PIN photodiodes (1223-01, Hamamatsu) in both integrating spheres were inputted into the control circuit, finally, sampled by PC. In this system, the reference measurements of laser power could reduce experimental error because of the fluctuance of laser power. The transmission intensity in the experiment was the ratio of the value detected by the big sphere to that by the reference sphere.²⁸

2.4.

Measurement Method

Experiments were conducted at room temperature $(\sim 25°\mathrm{C})$ . Each skin sample was clamped between two diffusion chambers, which were held together. $12\mathrm{ml}$ of OCAs was added to the donor cell in contact with the epidermal side of the skin, while $12\mathrm{ml}$ of isotonic saline was added to the receptor cell in contact with the dermal side. The chambers were set up with the receptor cell touching the port $(\varphi =25\mathrm{mm})$ of the integrating sphere. To determine the OCAs induced optical clearing of skin, the dynamical transmission intensity of samples was measured at 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, and $60\min$ after the application of OCAs. The relative transmittance can be deduced as the following formula:

After the measurement, the skin samples were taken from the chambers and wiped dry. The surrounding exposed area was cut away, and then the thickness of skin was measured.

2.5.

Statistical Analysis

SPSS 13.0 statistical software (SPSS Limited, Chicago, USA) was used to perform an F test among all the groups to compare the differences both in thickness changes and in relative transmittance after the experiment. Within each group, a paired-sample t-test was performed to compare skin thickness before and after an experiment. The Pearson correlation coefficient, relating relative transmittance to the refractive index and molecular weight of OCAs, was calculated.

3.1.

Relative Transmittance of Porcine Skin

Based on the measurements of transmission intensity, the relative transmittance of skin was deduced. Figure 2 shows the average changes of relative transmittance with time. The error bars in the plot are not shown because they are close in several groups.

Fig. 2

Relative transmittance of porcine skins after treatment with six alcohols and saline solution.

The results indicate that the relative transmittance of skins increases gradually after treatment with the OCAs. In contrast, there is some decrease in the control group alone after treatment with the isotonic saline. Among the alcohols, glycerol shows the greatest changes in relative transmittance, and 1-butanol shows the poorest.

3.2.

Skin Thickness and the Corresponding Change after the Experiment

Table 2 lists the thickness of samples before and after the experiment, and the corresponding change. It seems that there is slight shrinkage in all the experimental groups and a slight swell in controls. However, a paired-sample t-test shows that there is no significant difference within each group, and an F-test shows that there is no difference in thickness change among groups $(\mathrm{P}>0.05)$ .

Table 2

Skin thickness before and after experiments and the corresponding changes ( d0 is the thickness before experiments; d is the thickness after experiments; and Δd is the thickness changes after experiments).

3.3.

Correlation between the Optical Clearing Effect and the Number of Hydroxyl Groups in Chemical Structure

According to the number of hydroxyl groups in the chemical structure, we can divide the OCAs used in this work into three categories: monohydric alcohols (1-butanol), diatomic alcohols (PEG400, PEG200, 1, 3-propanediol, and 1, 4-butanediol), and triatomic alcohols (glycerol). The maximal relative transmittance of the skin $60\min$ after treatment with three categories of alcohols is shown in Fig. 3 . The maximum of the relative transmittance is almost 2.00 induced by glycerol, whereas the minimum is only 1.38 induced by 1-butanol. There is no statistical difference among the other four alcohols, diatomic alcohols, i.e., PEG400, PEG200, 1,3-propanediol, and 1,4-butanediol. Therefore, we can conclude the main features: 1. glycerol, triatomic alcohol, has the greatest effect; 2. 1-butanol, monohydric alcohol, has the poorest effect; and 3. the four diatomic alcohols are moderate. In other words, the more hydroxyl groups an OCA has in its chemical structure, the more effective it is in skin optical clearing.

Fig. 3

The maximal relative transmittance of skin samples treated by three categories of alcohols: triatomic, diatomic, and monohydric alcohols.

3.4.

Correlation between the Optical Clearing Effect and the Refractive Indices of Optical Clearing Agents

The refractive indices of the OCAs used in the work vary between 1.40 and 1.47. Figure 4 illustrates the relation between the maximal changes in the relative transmittance of skin and the refractive indices of OCAs. The results show that the correlation coefficient is only 0.12. To eliminate the effects caused by the chemical structure of OCAs, we further discussed diatomic alcohols’ induced optical clearing effect of skin. The correlation coefficient between their refractive indices and the corresponding maximal relative transmittance is 0.22. Therefore, the optical clearing effect of skin treated for $60\min$ has no correlation with the refractive index of OCAs.

Fig. 4

Correlation between the maximal relative transmittance of skin and OCA refractive indices.

3.5.

Correlation between Optical Clearing Effect and Molecular Weights of Optical Clearing Agents

The OCAs’ molecular weights and the corresponding relative transmittance of skin at $60\min$ are shown in Fig. 5 . The correlation coefficient is only 0.08. Though small substances $(<500\mathrm{Da})$ easily penetrate the skin, the molecular weights of alcohols do not affect the optical clearing of skin directly when they range from $74\text{to}400\mathrm{Da}$ .

Fig. 5

Correlation between the maximal relative transmittance of skin and OCA molecular weights.