Twenty-one female patients with alopecia areata, who came to Nagano Clinic (Tokyo, Japan) during the period from October 2014 to April 2015 were investigated in this study (age range 29 to 67 years; mean 45.0 ± 0.9 years). This study did not include any cancer patients and patients did not receive chemotherapy or irradiation therapy. Written informed consent was obtained from all subjects and the study was approved by the institutional review board of Nagano Clinic (Tokyo, Japan).

Images of tongues were captured by a digital image camera (cx3, Ricoh, Tokyo, Japan) before blood sampling at a fixed place in the medical examination room. Images were sent to experienced acupuncture therapists (>20 years experience) who were blinded to the patients. The acupuncture therapists categorized these images into a balanced group and unbalanced group. Then, the unbalanced group was divided into four groups based on the Eight Principles of TCM.

Venous blood (2 mL) was obtained from the forearm median antebrachial vein for analysis. Leukocyte number and subsets were determined using a hemocytometer and the May-Grünwald Giemsa staining method (Showa Medical Science Corporation, Tokyo, Japan). Thus, the total number of WBCs was examined. The proportion of granulocytes (including neutrophils, basophils, and eosinophil) and lymphocytes was determined.

The difference between the values of two groups (AT and AM) was determined by Student’s t-test, Mann- Whitney’s U-test and Welch’s t-test. Results are presented as the mean ± SD. P-values of < 0.05 were considered statistically significant, and all statistical tests were two-tailed.

Twenty-one female subjects of alopecia areata were divided into two groups by a dermatologist with 35 years of experience. The mean age of the AT group was 42 ± 8 years (n = 9) and was 46 ± 10 (n = 12) years in the AM group. There was no statistical difference between the two groups (Figures 1(A)-(C)).

Absolute numbers of WBCs of the two groups were compared (Figure 1(D)). WBC numbers were higher in the

AT group compared with the AM group (p < 0.05). The mean ratios of granulocytes and lymphocytes in the AT group was 64% ± 5% and 31% ± 5%, respectively, and were 61% ± 6% and 33% ± 7% in the AM group. There was no statistical difference in WBC subsets between the two groups (Figure 1(E) and Figure 1(F)).

The absolute number of granulocytes and lymphocytes were calculated as follows: (absolute number of WBCs in Figure 1(D)) × (percentage of granulocytes or lymphocytes (Figure 1(E) and Figure 1(F) respectively). Then the obtained numbers were statistically analyzed for each WBC subset (Figure 1(G) and Figure 1(H)).

The absolute number of granulocytes was higher in the AT group compared with the AM group (3982 ± 588 vs 3131 ± 836; p < 0.01). However, no difference was observed for lymphocytes between the two groups (1897 ± 275 vs 1667 ± 482).

The tongue images were categorized into five groups: balanced [normal] group and four unbalanced groups: Yin+ (Shihan, excess coldness), Yang− (Xuhan, deficit heat), Yin− (Xure, deficit coldness) and Yang+ (Shire, excess heat) groups (Figure 2).

In the AT group the ratios of those groups were 11%, 56%, 0%, 33%, and 0% (normal, Yin+, Yang−, Yin− and Yang+), respectively. In the AM group they were 0%, 42%, 8%, 42% and 8% respectively (Figure 3(A) and Figure 3(B)).

The ANS contains two types of nerves: sympathetic nerves, which are dominant when we suffer stress and parasympathetic nerves that are dominant when we relax. They maintain our health by balancing each other [7] (Figure 2(A)). This balance is easily altered by small changes in our health. For example, the duration of sleep can affect this balance [8] [9] . Furthermore, long-lasting stress induces unbalanced internal conditions that might

be involved in the onset of various diseases [10] . Because the ANS controls the distribution of WBCs, the status of ANS can be assessed by investigating WBC subsets [1] - [3] .

Our results of WBC subsets showed that the sympathetic nerves were more dominant in the AT group compared to the AM group because the AT had higher numbers of granulocytes than the AM group (Figure 1(G)). Because the number of granulocytes was calculated based on the WBC numbers the difference in WBCs between two groups can be understood in the same way as granulocytes (Figure 1(D)).

Alopecia is the loss of hair. Patients with multiple areas of alopecia lesions are termed AM and patients who lose all the hair on the scalp are termed AT [11] . There are several theories to explain this condition, including the increase of natural killer (NK) cells in the peripheral blood lymphocytes of patients [12] [13] .

Our previous study indicated that under sympathetic nerve dominance, the number, ratio and function of NK cells were enhanced [14] [15] . This is explained by two points. First, sympathetic nerves elevate the level of adrenaline [14] - [16] and second, NK cells express adrenaline receptors [17] - [19] . Interestingly, granulocytes also mainly express adrenergic receptors (lymphocytes express cholinergic receptors on the cell surface) [20] and that granulocytes are stimulated by sympathetic nerves (lymphocytes are stimulated by parasympathetic nerves) (Figure 2). Thus, the prominent differences between the number of WBCs and granulocytes between two groups might indicate patients of those two groups have different ANS balances.

Recently we reported that repetitive manual acupuncture increased markers of innate immunity in mice subjected to restraint stress [23] . This result might agree with the description in TCM, “Huangdi Neijing”, because, anger stress might influence the liver.

In addition, we found that acupuncture stimuli were effective only within the range of normal homeostasis [23] . Therefore, in general, statistical differences in the results of alternative medicine (such as acupuncture stimuli) might not be as prominent as those of conventional drugs (such as anti-inflammatory agents, fever reducers, or diabetes drugs). For example, our results of the tongue inspection suggested a certain degree of consistence with those of WBC analysis. Moreover, some subjects in the Yin+ and Yang+ groups showed clearer results in the tongue inspection compared to the WBC subsets although no statistical differences were observed (data not shown). Therefore, traditional techniques such as tongue inspection and acupuncture must be studied to detect subtle effects induced by acupuncture treatment. Thus, a comprehensive approach is needed at the bedside because the tongue inspection only one part of the “four inspections” in TCM (inspection, listening/smelling, inquiring and palpation).

Our study has several limitations. This study may have been underpowered to detect small differences, and further research with larger scale studies including males is required. Moreover, we should obtain samples from healthy subjects to compare with those of the AT and AM groups. Additionally, body temperature and pulse rates obtained by western methods should be assessed as we studied the effect of an ancient type of acupuncture needle [24] -[26] . If we could obtain those data in larger samples, multiple logistic regression analysis could be used to investigate the relationship between the tongue inspection and clinical assays of WBC subsets to contribute to medical advances in the future.