It is known that acupuncture stimulation affects blood
flow, and there are some studies for skin (1), muscle (2)
and brain (3). We examined whether acupuncture not
only stimulates a local area but also the blood flow of
other organs, thus clarifying how acupuncture stimulates
an organism. Since it is necessary to examine the effect
on organ blood flow according to different areas of
stimulation, the blood flow of various organs was
measured in anesthetized rats using colored microspheres
that can quantitatively measure multiple organ blood
flow. We examined how acupuncture stimulation of the
regions (Hsia-Kuan or Hoku) influenced the blood
flow of various organs. Although microsphere measure-
ment for regional blood flow has radiolabeled micro-
spheres (4-6,7-17,18) and colored microspheres (19-28),
we used the colored ones in this experiment. There are
two techniques for colored microsphere measurement.
One technique can calculate blood flow by counting the
total number of microspheres in each sample (19-23)
and the other by extracting colored dye from the
microsphere s (24-28). We used the latter technique in
our experiment.
After 24 h without food, Male Wistar rats (n ¼ 27, body
weight: 250-420 g, free water intake) were anesthetized
with intra-peritoneal injection (1.2 g/kg) of urethane.
After tracheotomy, a cannula was inserted and a
respirator artificially regulated breathing (respiratory
frequency: 90 cycles/min, tidal air: 10 ml/kg, SN-480-7,
Shinano, Japan). Pancuronium bromide (2 mg/kg) was
administrated from a catheter that was placed in the
jugular vein of rats. In addition, CO 2 concentration in
the expiration was monitored (1H26, NEC) and main-
tained at about 3%. A second catheter (PE-50) was
positioned in the right femoral artery to monitor blood
pressure. The blood pressure and heart rate were
recorded on a thermal array recorder (RTA-1200,
Nihon Kohden). A third catheter (PE-10) was inserted
into left ventricular via the right carotid artery for the
colored microsphere injection. And, a fourth catheter
(PE-50) was positioned in the left femoral artery for
withdrawal of blood samples by a syringe pump at a rate
of 0.84 ml/min (Model210, KD Scientific Inc. USA). The
rectal temperature was monitored using a thermistor and
maintained about 37.5 C by means of a heating pad
(MK-900, Muromachi Kikai Co.). In this experiment,
yellow and blue microspheres (15 AE 0.2 mm, Dye-Track
Triton Technology Inc. USA) were used to measure
organ blood flow.
The position of the left ventricle catheter was confirmed
by autopsy at the end of experiment.
The infusion of colored microspheres started at least
60 min after surgery and confirmation of stabilized
blood pressure and heartbeats. The microspheres were
stirred with a test tube mixer (NS-80, Iuchiseieidou) for
5 min before infusion. The reference blood was drawn
from 10 s before the microsphere infusion, and continued
for 75 s. The microsphere (yellow or blue) infusion
(20 s) was started 10 s after beginning to draw blood.
Saline (0.5 ml) was then infused for 30 s to flush the
microspheres in the catheter. In all experiments, yellow
microspheres (0.12-0.14 ml, 360 000-420 000 micro-
spheres) were injected first and blue (0.2-0.23 ml,
600 000-690 000 microspheres) ones second. After yellow
injection, additional fluid was not replaced except by
injection of blue. The injection of blue microspheres
started 30 min after the first blood sampling was finished
in the control group. In ST-7 or LI-4 group, acupuncture
stimulation was applied after the first sampling. About
30 min after inserting the acupuncture needle, blue micro
spheres were injected.
Figure 1 shows examples of the absorbance by the
above processing. Since the peak of absorbance in yellow
microspheres appears at 448 nm wavelength and blue
appears at 672 nm (24,27), we measured 448 nm for
yellow microspheres in the blood sample of and 672 nm
for blue microspheres. Tissue samples containing both
microspheres were measured at wavelength absorbencies
of 448 nm and 672 nm. Tissue samples with no absor-
bency peak were deleted from our data.
Organ blood flow was calculated using the equation
below:
Qm shows blood flow of the tissues (ml/min/g). Qr shows
the withdrawal rate of the blood samples. Am shows the
absorbance (AU) of the microspheres per 1 g. Ar shows
the absorbance (AU) of all microspheres in the blood
samples.
Data were expressed as the mean AE SD. The percentage
changes of blood flow were expressed as 100% the first
value of blood flow, and the percentage was showed
by box and whisker plot. Wilcoxon signed rank test,
Mann-Whitney U-Test, One-way or Two-way ANOVA
was used for data analysis. Differences of P50.05 were
considered statistically significant.
Figure 1 shows the time course of mean blood pressure
(mmHg) in the control, ST-7 and LI-4 group. The mean
blood pressures before the first withdrawal in the control,
ST-7 and LI-4 were 76.0 AE 6.0, 80.7 AE 12.7 and
86.4 AE 11.7. Although blood pressure of the control
group
tended
to
be
low,
there
was
no
significant difference among the three groups (P ¼ 0.15).
One-way ANOVA was applied to this analysis. The
temporal changes of blood pressure were also similar
among three groups, and no significant differences
(F(2,14) ¼ 1.94, P ¼ 0.17) and interaction (P ¼ 0.69)
among three groups. On the other hand, heart rate
(beats/min) before the first withdrawal of the control
group, ST-7 and LI-4 were 383.7 AE 25.3, 424.6 AE 40.3 and
427.4 AE 27.6.There was no significant difference
[F(2,14) ¼ 3.02, P ¼ 0.07] and interaction (P ¼ 0.45)
among the three groups (data not shown). Two-way
ANOVA was applied to these analyses.
The second measurements of organ blood flow were
slightly lower than those of the first in every organ, with
significant differences in the muscle, kidney, stomach,
brain and spleen (Fig. 2). Wilcoxon signed rank test
was applied to these analyses and the mean variations
(ml/min/g) of first and second organ blood flow in each
organ were as follows; kidney: À0.65; small intestine:
À0.49; lung: À0.33; spleen: À0.32; stomach: À0.26; brain:
À0.17; muscle: À0.05; heart: À0.03 and liver: À0.02.
The first and second blood flow of the left masseter
muscle in the control group were 0.35 AE 0.24, 0.36 AE 0.45,
respectively (P ¼ 0.35, no figure), and right masseter
of the control group were 0.16 AE 0.18 and 0.12 AE 0.10,
and there was no significant difference (P ¼ 0.34,
no figure).
Figure 3 shows the first and second organ blood flows in
the ST-7 group. Though the second blood flow was
slightly higher than the first blood flow in the muscle,
stomach, small intestine, brain and heart, there was
Mean percentage change of the left masseter stimulated
by acupuncture in the ST-7 group was þ57.2. The value
of the right masseter in the ST-7 group that was not
stimulated was þ28.9. The control group values were:
left masseter: À10.8; right masseter: À11.3. While
the blood flow rate decreased in both masseters of
the control group, the blood flow rate of the left
masseter of the ST-7 group had increased more than
the right of the same group. However, this difference
between the left and right masseters of the ST-7
group was not statistically significant. (Mann-Whitney
U-test).
Figure 5 shows the first and second organ blood flow
measurements of the LI-4 group. Though the second
blood flow increased slightly more than the first in the
brain, lung and heart, there was no significant difference.
There was a significant decrease in the muscle. Wilcoxon
signed rank test was applied to these analyses. Mean
variation (ml/min/g) of blood flow of each organ blood
flow was: heart: þ2.15; lung: þ0.16; brain: þ0.11; liver:
0.00; stomach: À0.01; muscle: À0.05; small intestine:
À0.18; spleen: À0.54 and kidney: À0.90. Figure 6 shows
the percentage change (%) of the control group and LI-4
group by box and whisker plot. There was no significant
difference between the control group and LI-4 group.
Mann-Whitney U-test was applied to these analyses.
Mean percentage change (%) of organ blood flow in LI-4
group were described subsequently; lung: þ46.3; heart:
þ34.7; brain: þ11.0; liver: þ7.4; small intestine: À1.1;
stomach: À10.8; kidney: À16.9; muscle: À30.0 and spleen:
À33.3.
The colored microsphere technique used in this experi-
ment has various advantages for organ blood flow
measurement. It can measure blood flow of multiple
organs simultaneously. In principle, microspheres are
trapped at the peripheral capillary, and when infusion
volume increases, the measurement accuracy will rise
(18). However, disturbances may occur in the rat's
circulation. Kobayashi et al. (22) described that a bolus
injection of less than one million colored microspheres
caused no significant hemodynamic disturbances in rats,