J Acupunct Meridian Stud 2010;3(1):4952

B RI E F R EP ORT

Visualization of Acupuncture Meridians in the

Hypodermis of Rat Using Trypan Blue
Byung-Cheon Lee1,2*, Kwang-Sup Soh1
1

Biomedical Physics Laboratory, Department of Physics and Astronomy, Seoul National University,
Seoul, Korea
2
Pharmacopuncture Medical Research Center, Korean Pharmacoacupuncture Institute, Seoul, Korea

Received: Oct 6, 2009

Accepted: Dec 3, 2009
KEY WORDS:
acupuncture;
meridian;
primo-node;
primo-vessel;
Trypan blue

Abstract
Bonghan Kim claimed that the primo-vessel (Bonghan duct) and the primo-node
(Bonghan corpuscle) are anatomical structures corresponding to acupuncture meridians and acupoints, respectively, but this claim has not been confirmed. Recently
we found that Trypan blue preferentially stained primo-vessels and primo-nodes on
the surfaces of internal organs, not staining other structures like blood or lymph
vessels or nerves. In this work, we applied Trypan blue to visualize the putative
acupuncture meridian along skin skeletal muscles in the hypodermal layer of a rat.
The Trypan blue stained-structures are morphologically similar to the meridian
primo-vessels, as claimed by Bonghan Kim. Further study is needed to investigate
the network of these Trypan blue-stained structures in order to establish them as
acupuncture meridians.

1. Introduction
In the 1960s, Bonghan Kim claimed that acupuncture
meridians were anatomical structures named meridian primo-vessels (superficial Bonghan ducts) [1].
Over several years we have conducted investigations
to rediscover primo-vessels (Bonghan ducts) and
primo-nodes (Bonghan corpuscles) in various tissues
of mouse, rat, and rabbit [210]. In addition to these
anatomical observations we found catecholamines
in the primo-fluid (Bonghan liquid) extracted from
primo-nodes, and we measured the flow speed of
the primo-fluid [11,12]. Additionally, we performed
proteomics analysis on the primo-fluid extracted
from the primo-vessels of rabbit [13]. We have
very recently found an effective dye, Trypan blue,

which strongly and preferentially stained the primovessels on internal organs and other tissues [14,15].
In this work, using Trypan blue, we were able to
visualize novel muscles containing sinuses that lay
in parallel with skin skeletal muscles in the hypodermal layer of a rat. We observed several features
of the Trypan blue stained structures (TBS) that
were similar to the characteristics of the meridian
primo-vessels described by Bonghan Kim [1].

2. Materials and Methods

Rats (Wistar/ST males, 200 g; Jung-Ang Laboratory
Animal Co., Seoul, Korea) were housed at 23C and
60% relative humidity under a 12-hour light/dark

*Corresponding author. Biomedical Physics Laboratory, Department of Physics and Astronomy, Seoul National University, Seoul,
151-747, Korea.
E-mail: donboscolee@gmail.com
2010 Korean Pharmacopuncture Institute

50

B.C. Lee, K.S. Soh

cycle with ad libitum access to food and water.

Animals were handled in accordance with the
Guidelines of the Laboratory Animal Care Advisory
Committee of Seoul National University. Under urethane (1.5 g/kg) anesthesia, we dissected the medial alba of a rat to expose the subcutaneous layer.
The exposed subcutaneous area was stained with
0.4% Trypan blue (Sigma-Aldrich, St-Louis, MO, USA)
and washed with saline two or three times. The TBS
along the skin skeletal muscle was observed and
pictures were taken under a stereomicroscope using
a CCD camera (SZX 12 with DP 70; Olympus, Tokyo,
Japan).
We isolated the TBS for light microscopic examination. For the histological study, the TBS-containing
skin was fixed with 10% neutral buffered formalin.

After fixation, we made paraffin blocks of the TBScontaining skin and sliced 5-m sections from
these blocks for hematoxylin and eosin staining.

3. Results
Figure 1 shows images of the meridian primo-vessel
(superficial Bonghan duct) in the hypodermal layer,
which were taken by Bonghan Kim [1]. Figure 1B,
a magnified view of a part of Figure 1A, shows the
presence of slits which are indicated by arrows.
Figure 2A shows an image of the hypodermal layer
before staining with Trypan blue. After staining
with Trypan blue, a straight blue line, which is indicated by arrows, emerges as shown in Figure 2B.

(Meridian)
Figure 1 (A) Light microscopic image of a meridian primo-vessel (superficial Bonghan duct) reported by Bonghan
Kim [1]. (B) Magnified view of (A). The slits indicated by arrows are a noticeable feature. A primo-vessel is composed
of many channels like a vascular bundle. This original picture had no information on the scale bar and staining name.

H D

Hair
200 m

200 m

SSM

200 m

Figure 2 Stereoscopic images of skin that is (A) without staining, (B) with Trypan blue (TB)-staining and (C) magnified view of (B). TB-stained subcutaneous skin (B) reveals a blue-colored threadlike structure, part of which is magnified in (C). Notice that the skin skeletal muscle (SSM) just near the blue line was not stained by TB. H = hypodermis;
D = dermis.

Visualization of acupuncture meridians using Trypan blue

A magnified view of this line in which the putative
primo-vessel is seen as a distinctive blue line (arrows) just beside the skin skeletal muscle (SSM),
can be seen in Figure 2C.
Figure 3A presents a light microscopic image of
the TBS which had some slits (arrows) similar to
those in the image in Figure 1B. Figure 3B shows a
magnified view of the dotted rectangle in Figure 3A,
which shows the difference between the TBS and
the SSM. The SSM has distinctive striated lines, which
are indicated by arrows; however, the TBS has no
striated lines. After the TBS had been cut, its tip was
a little shrunken and round in shape (see the solid

51

circle in Figure 3B), but the tip of SSM tip was sharply
cut (see the dotted circle in Figure 3B). Figure 4A
presents cross-sectioned images of the TBS and the
SSM. The TBS is magnified in Figure 4B, which shows
small or big-sized sinuses, which are indicated by
arrows and dotted arrows, respectively.

4. Discussion
The TBSs we found in the hypodermis of rat have
features similar to those of primo-vessels (superficial Bonghan ducts). These include the preferential

100 m

20 m

Figure 3 A piece of skin tissue in Figure 2 taken and observed with a light microscope. (A) Microscopic image of a
Trypan blue-stained structure (TBS) and nearby skin skeletal muscle (SSM) unstained by Trypan blue. The TBS has
noticeable slits, which are indicated by arrows. (B) The dotted rectangle in (A) is magnified. Nuclei are shown blue by
DAPI. The SSM has distinctive striated lines on its surfaces whereas the TBS does not. The slit in the TBS is indicated by
an arrow. Also, a noticeable difference between the circle (tip of the TBS) and the dotted circle (tip of the skin skeletal
muscle) is apparent. After simultaneous cutting of the TBS and the SSM had occurred, the tip of the TBS (the lined
circle) was a little shrunken and slightly rounded in shape, but the tip of the SSM (the dotted circle) was cut sharply.

100 m

20 m

Figure 4 Hematoxylin and eosin staining of the cross sections of skin skeletal muscle (SSM) and Trypan blue-stained
structures (TBS). (A) Cross section of skin containing the SSM and the TBS. (B) Magnified view of the TBS from the dotted rectangle of Figure 4A in which there are small sinuses (dotted arrows) and large sinuses (solid arrows).

52

B.C. Lee, K.S. Soh

staining by Trypan blue [14], the bundle of musclelike structures and their thickness (about 150 m)
[2], and the presence of sinuses of various sizes in
the muscle-like structures [4,14]. The sinuses are
strong evidence that the TBSs are channels where
fluid flows, much like the primo-vessels [12]. Finally, we note that there are slits in the TBS (Figure
3A) as in the meridian primo-vessel of Kim (Figure
1B). This kind of slit was also noticed in the primovessel observed in the brain ventricles of a rabbit
[3]. The presence of slits suggests that the TBS may
have a contractile property to transport fluid in
the channels depicted by the sinuses.
Even though we found the TBS along the skeletal muscle in the hypodermis of the abdominal
skin of a rat, the success rate was low because the
staining technique was difficult. The putative acupuncture meridian is thought to form a network
that has not yet been found by using the current
method. According to Bonghan theory [1], an acupuncture point is a rather thick and bulged body
connected to a meridian which has not yet been
observed in our work. Further work and improvement of technique is needed to establish the whole
primo-vessels and primo-nodes as anatomical structures corresponding to acupuncture meridians and
acupuncture points.

3.

4.

5.

6.

7.

8.

9.

10.

11.

Acknowledgments
This work was supported by a Systems Biology
Infrastructure Establishment Grant provided by
the Gwangju Institute of Science and Technology
in 2009.

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