Twenty-five healthy male volunteers, 24 to 60 years of age, participated in this study. Written informed consent was obtained from all subjects and the study was approved by the institutional review board of Niigata University.

The DZ needles were made of stainless steel and had a column-like body and a flat head (Soken Medical Co. Ltd., Toshima, Japan). An autoclaved DZ was softly pressed vertically onto the body surface for 5 minutes (diameter 5.0 mm, 37.3 g/mm²) (Figure 1(a)), at approximately 120 points from the head to the feet on both sides of the spine (Figure 1(b)). The DZ application points also corresponded to the Bladder Meridian as defined by the World Health Organization [3]. The treatment was applied by nationally licensed experienced Japanese acupuncturists.

For measurement of body temperature and pulse rate before and after DZ treatment, the subjects were divided randomly into two age-matched groups. Half of the subjects (n = 13; 37.4 ± 12.6 years) received DZ application and the matched controls (n = 12; 37.8 ± 13.7 years) received no treatment. For measurements other than body temperature and pulse rate, six randomly selected subjects (34.5 ± 10.9 years) received DZ treatment and were compared to six age-matched controls (33.1 ± 14.3 years).

Core temperature (sublingual) was measured with a clinical mercury thermometer (FAVOR, Nihon Keiryoki Kogyo Co. Ltd., Chiyoda, Japan) before and after DZ application. The cutaneous body temperature was measured using a contactless digital thermometer (Microlife, IR 1DA1, microlife, Switzerland) before and after DZ at five locations: Yintang (on the forehead, mid-way between the medial ends of the eyebrows), LI4 (Hegu; on the dorsum of the right and left hands, radial to the mid-point of the second metacarpal bone), LR3 (Taichong; on the

dorsum of the right and left feet, between the first and second metatarsal bones, in the depression distal to the junction of the bases of the two bones, over the dorsalis pedis artery) (Figure 1(c)).

Fresh venous blood was obtained before and after the DZ application for the measurement of PO₂, SO₂, PCO₂ and hemoglobin, using i-STAT 300 F (i-STAT Corporation, East Windsor, NJ, USA) as previously described [4-7]. WBCs were measured using a hemocytometer and the May-Grünwald Giemsa stain method. Concentrations of catecholamines (adrenaline, noradrenaline and dopamine) were measured by high-performance liquid chromatography (SRL, Inc., Tokyo, Japan). Additionally WBCs and catecholamines were examined at one week after DZ application.

The statistical significance of differences between values was tested using paired t-tests or repeated-measures two-factor ANOVA and a Bonferroni/Dunn post-hoc test. P values of less than 0.05 were considered to be statisticcally significant.

Pulse rate and sublingual body temperature were measured before and soon after DZ application. The pulse rate showed a prominent decrease after DZ application (from 71.4 ± 4.7 to 66.2 ± 5.4 /min, P < 0.05), but the body temperature did not change significantly (36.8 ± 0.3 versus 36.6˚C ± 0.4˚C, P > 0.05). No changes were seen in the control group (Figure 2(a)).

Because a drop in pulse rate might affect blood circulation and thereby cause a decrease in the oxygen content of venous blood, we measured oxygen and carbon dioxide pressure (PO₂ and PCO₂) and oxygen saturation (SO₂). We found that PO₂ and sO₂ both decreased significantly after DZ application (28.9 ± 10.9 to 19.4 ± 10.5 mmHg, P < 0.05; and 46.8% ± 9.4% to 24.8% ± 9.1%, P < 0.05, respectively). These changes tended to recover within 60 minutes after DZ application; however, this apparent return to baseline was not statistically significant owing to high inter-subject variability (data not shown). Neither PCO₂ nor Hb showed any prominent changes. In the control group, no changes were observed in any of the parameters (Figure 2(b)).

It has been reported that rapid changes in core temperature is associated with changes in cutaneous blood flow mediated by the autonomic nervous system (ANS) [8]. Although it was not statistically significant DZ application induced a tendency of a rapid decrease in core temperature (Figure 2(a)). Therefore, we measured cutaneous body temperature over time at five points (Figure 1(c)). Soon after DZ stimulation, the temperature of the hands and feet appeared to transiently decrease (average: hands, △0.7˚C ± 0.6˚C; feet, △1.9˚C ± 1.5˚C) then began to recover after 20 minutes. However, these

changes were not statistically significant (P > 0.05) (Figure 3).

The levels of catecholamines are known to reflect the status of the ANS [9-11]; thus, these were measured before and after the DZ application. We found that the serum levels of adrenaline and noradrenaline were significantly elevated after DZ treatment (from 33 ± 7 to 39 ± 10 pg/mL, P < 0.05; and from 216 ± 69 to 306 ± 92 pg/mL, P < 0.05; respectively). Dopamine did not change significantly (Figure 4(a)). One week after DZ application, there was no statistically significant difference in catecholamine levels compared to baseline (data not shown).

The above finding of an elevation in adrenaline and noradrenaline indicates dominance of sympathetic nervous activity. Mori et al. reported that electric acupuncture affected ANS status and had an immunomodulatory effect [9]. To investigate the effect of DZ application on immune function and ANS status the number and ratio of WBCs were studied.

After DZ application the ratio of granulocytes appeared to increase (66% ± 6% to 61% ± 9%, P > 0.05) while that of lymphocyte tend to decrease though they were not

statistically significant (33% ± 6% to 37% ± 8%, P > 0.05) (Figure 4(b)). The absolute numbers of granulocytes and lymphocytes were determined by calculation. The number of lymphocytes showed a step-like elevation and there was a statistically significant difference between levels at baseline versus at one week after treatment (2159 ± 90 to 2485 ± 51 /μL) (P < 0.05) (Figure 4(b)).

In a series of previous studies, we have found body temperature to be an important parameter in understanding metabolic conditions induced by changes in ANS activity and immune status [6,10,11]. The sublingual and cutaneous temperature decreased after DZ application though they were not statistically significant (Figures 2(a) and 3), while the levels of adrenaline and noradrenaline increased (Figure 4(a)). Because adrenaline and noradrenaline are known to be elevated under conditions of sympathetic dominance [11,12], it is likely that the observed elevation in catecholamines reflect an increase in sympathetic activity induced by DZ application. Adrenaline α-receptors are involved in mediating decreases in body temperature [11]. It has been reported that adrenaline β-receptors mediate the constriction of heart or vascular smooth muscle and decreases in pulse rate caused by acupuncture via ANS modulation [9,13]. Our results are consistent with these previous reports.

A continuous state of high sympathetic activity induces adaptive responses to maintain homeostasis [10,11, 14,15]. However, the responses caused by DZ application were transient, with recovery of the cutaneous hypothermia seen within 20 minutes (Figure 3). In this pilot study, we confirmed that the decreased pulse rate and sublingual hypothermia also showed recovery within 20 to 40 minutes, and that the levels of catecholamines recovered the following week. While the great inter-individual variability prevented firm conclusions, the changes in all subjects showed the same trend toward recovery (data not shown). This supports the hypothesis that the changes in sympathetic status induced by DZ application were transient.

DZ application induced a rapid change in the WBC profile. The number of lymphocytes did not change on the day of the application but showed a step-like elevation after one week (Figure 4(b)). Thus it is undeniable that DZ application affected the ratio and number of WBCs via ANS modulation, as previously reported [16].

We recently investigated the effect of skin rubdown with a dry towel (SRDT), which stimulated almost the same part of the skin [4]. Interestingly, DZ application and SRDT induced precisely the opposite effects on pulse rate and sublingual body temperature, even though they are both considered to affect the immune system via ANS modulation. One reason may be found in the difference of the stimuli applied with the two methods. DZ application stimulated a very narrow point, while SRDT mildly stimulated a wider area of the skin. The effect of DZ application was much stronger than that of SRDT. In fact, the changes in adrenaline level and noradrenaline level after DZ application were 1.2 times and 1.3 times greater than those of SRDT [4]. This might indicate that these two treatments applied different doses of stimulation and that the dose of the DZ application was stronger than that of SRDT. Another reason may be differences in the location of the stimulated tissue. It is possible that stimulation with stronger pressures may stimulate deeper components of the tissue. Last, a possible reason might be the compatibility of the ancient type of acupuncture needle with the stimulated acupuncture points along the Bladder Meridian. If this historically reported compatibility is significant in the effect of DZ compared to other treatments, it should be possible to observe effects that are specific for DZ application. Further research is required to support this hypothesis.