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.
Brief Report

Visualization of Acupuncture Meridians in the Hypodermis of Rat Using Trypan Blue
aBiomedical Physics Laboratory, Department of Physics and Astronomy, Seoul National University, Seoul, Korea
bPharmacopuncture Medical Research Center, Korean Pharmacoacupuncture Institute, Seoul, Korea
2010; 3(1): 49-52
Published March 1, 2010 https://doi.org/10.1016/S2005-2901(10)60008-8
Copyright © Medical Association of Pharmacopuncture Institute.
Abstract
Keywords
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 [2, 3, 4, 5, 6, 7, 8, 9, 10]. 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 primo-vessels 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 23°C and 60% relative humidity under a 12-hour light/dark cycle with
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 TBS- containing skin and sliced 5-mm 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.
-
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.
-
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.
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 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.
-
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.
-
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).
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 staining by Trypan blue [14], the bundle of muscle- like structures and their thickness (about 150 mm) [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 primo- vessel 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.
Acknowledgments
This work was supported by a Systems Biology Infrastructure Establishment Grant provided by the Gwangju Institute of Science and Technology in 2009.
References
- Kim BH. On the Kyungrak System.
J Acad Sci DPR Korea 1963:1-35. - Shin HS, Johng HM, Lee BC, Cho SI, Soh KS, Baik KY, et al. Feulgen reaction study of novel threadlike structures (Bonghan ducts) on the surfaces of mammalian organs.
Anat Rec B New Anat 2005;284:35-40. - Lee BC, Kim S, Soh KS. Novel anatomic structures in the brain and spinal cord of rabbit that may belong to the Bonghan system of potential acupuncture meridians.
J Acupunct Meridian Stud 2008;1:29-35. - Lee BC, Yoo JS, Ogay V, Kim KW, Dobberstein H, Soh KS, et al. Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs.
Microsc Res Tech 2007;70:34-43. - Lee BC, Baik KY, Johng HM, Nam TJ, Lee J, Sung B, et al. Acridine orange staining method to reveal the characteristic features of an intravascular threadlike structure.
Anat Rec B New Anat 2004;278:27-30. - Lee BC, Yoo JS, Baik KY, Sung B, Lee J, Soh KS. Development of a fluorescence stereomicroscope and observation of Bong-Han corpuscles inside blood vessels.
Indian J Exp Biol 2008;46:330-5. - Yoo JS, Kim MS, Ogay V, Soh KS. In vivo visualization of Bonghan ducts inside blood vessels of mice by using an Alcian blue staining method.
Indian J Exp Biol 2008;46:336-9. - Lee BC, Yoo JS, Baik KY, Kim KW, Soh KS. Novel threadlike structures (Bonghan ducts) inside lymphatic vessels of rabbits visualized with a Janus Green B staining method.
Anat Rec B New Anat 2005;286:1-7. - Lee C, Seol SK, Lee BC, Hong YK, Je JH, Soh KS. Alcian blue staining method to visualize Bonghan threads inside large caliber lymphatic vessels and X-ray microtomography to reveal their microchannels.
Lymphat Res Biol 2006;4:181-90. - Johng HM, Yoo JS, Yoon TJ, Shin HS, Lee BC, Lee C, et al. Use of magnetic nanoparticles to visualize threadlike structures inside lymphatic vessels of rats.
Evid Based Complement Alternat Med 2007;4:77-82. - Kim JD, Ogay V, Lee BC, Lee BC, Kim MS, Lim I, et al. Catecholamine producing novel endocrine organ: Bonghan system.
Med Acupunct 2008;20:97-102. - Sung B, Kim MS, Lee BC, Yoo JS, Lee SH, Kim YJ, et al. Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs.
Naturwissenschaften 2008;95:117-24. - Lee SJ, Lee BC, Nam CH, Lee WC, Jhang SU, Park HS, et al. Proteomic analysis for tissues and liquid from Bonghan ducts on rabbit intestinal surfaces.
J Acupunct Meridian Studies 2008;1:97-109. - Lee BC, Kim KW, Soh KS. Visualizing the network of Bonghan ducts in the omentum and peritoneum by using Trypan blue.
J Acupunct Meridian Studies 2009;2:66-70. - Lee BC, Bae KH, Jhon GJ, Soh KS. Bonghan system as mesenchymal stem cell niches and pathways of macrophages in adipose tissues.
J Acupunct Meridian Studies 2009;2:79-82.
Related articles in JAMS

Article
Brief Report
2010; 3(1): 49-52
Published online March 1, 2010 https://doi.org/10.1016/S2005-2901(10)60008-8
Copyright © Medical Association of Pharmacopuncture Institute.
Visualization of Acupuncture Meridians in the Hypodermis of Rat Using Trypan Blue
Byung-Cheon Leeab*, Kwang-Sup Soha
aBiomedical Physics Laboratory, Department of Physics and Astronomy, Seoul National University, Seoul, Korea
bPharmacopuncture Medical Research Center, Korean Pharmacoacupuncture Institute, Seoul, Korea
Correspondence to:Byung-Cheon Lee
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.
Keywords: acupuncture, meridian, primo-node, primo-vessel, Trypan blue
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 [2, 3, 4, 5, 6, 7, 8, 9, 10]. 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 primo-vessels 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 23°C and 60% relative humidity under a 12-hour light/dark cycle with
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 TBS- containing skin and sliced 5-mm 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.
-
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.
-
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.
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 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.
-
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.
-
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).
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 staining by Trypan blue [14], the bundle of muscle- like structures and their thickness (about 150 mm) [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 primo- vessel 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.
Acknowledgments
This work was supported by a Systems Biology Infrastructure Establishment Grant provided by the Gwangju Institute of Science and Technology in 2009.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

There is no Table.
References
- Kim BH. On the Kyungrak System.
J Acad Sci DPR Korea 1963:1-35. - Shin HS, Johng HM, Lee BC, Cho SI, Soh KS, Baik KY, et al. Feulgen reaction study of novel threadlike structures (Bonghan ducts) on the surfaces of mammalian organs.
Anat Rec B New Anat 2005;284:35-40. - Lee BC, Kim S, Soh KS. Novel anatomic structures in the brain and spinal cord of rabbit that may belong to the Bonghan system of potential acupuncture meridians.
J Acupunct Meridian Stud 2008;1:29-35. - Lee BC, Yoo JS, Ogay V, Kim KW, Dobberstein H, Soh KS, et al. Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs.
Microsc Res Tech 2007;70:34-43. - Lee BC, Baik KY, Johng HM, Nam TJ, Lee J, Sung B, et al. Acridine orange staining method to reveal the characteristic features of an intravascular threadlike structure.
Anat Rec B New Anat 2004;278:27-30. - Lee BC, Yoo JS, Baik KY, Sung B, Lee J, Soh KS. Development of a fluorescence stereomicroscope and observation of Bong-Han corpuscles inside blood vessels.
Indian J Exp Biol 2008;46:330-5. - Yoo JS, Kim MS, Ogay V, Soh KS. In vivo visualization of Bonghan ducts inside blood vessels of mice by using an Alcian blue staining method.
Indian J Exp Biol 2008;46:336-9. - Lee BC, Yoo JS, Baik KY, Kim KW, Soh KS. Novel threadlike structures (Bonghan ducts) inside lymphatic vessels of rabbits visualized with a Janus Green B staining method.
Anat Rec B New Anat 2005;286:1-7. - Lee C, Seol SK, Lee BC, Hong YK, Je JH, Soh KS. Alcian blue staining method to visualize Bonghan threads inside large caliber lymphatic vessels and X-ray microtomography to reveal their microchannels.
Lymphat Res Biol 2006;4:181-90. - Johng HM, Yoo JS, Yoon TJ, Shin HS, Lee BC, Lee C, et al. Use of magnetic nanoparticles to visualize threadlike structures inside lymphatic vessels of rats.
Evid Based Complement Alternat Med 2007;4:77-82. - Kim JD, Ogay V, Lee BC, Lee BC, Kim MS, Lim I, et al. Catecholamine producing novel endocrine organ: Bonghan system.
Med Acupunct 2008;20:97-102. - Sung B, Kim MS, Lee BC, Yoo JS, Lee SH, Kim YJ, et al. Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs.
Naturwissenschaften 2008;95:117-24. - Lee SJ, Lee BC, Nam CH, Lee WC, Jhang SU, Park HS, et al. Proteomic analysis for tissues and liquid from Bonghan ducts on rabbit intestinal surfaces.
J Acupunct Meridian Studies 2008;1:97-109. - Lee BC, Kim KW, Soh KS. Visualizing the network of Bonghan ducts in the omentum and peritoneum by using Trypan blue.
J Acupunct Meridian Studies 2009;2:66-70. - Lee BC, Bae KH, Jhon GJ, Soh KS. Bonghan system as mesenchymal stem cell niches and pathways of macrophages in adipose tissues.
J Acupunct Meridian Studies 2009;2:79-82.