Novel threadlike structures (Bonghan ducts) inside lymphatic vessels of rabbits visualized with a Janus Green B staining method

Authors

  • Byung-Cheon Lee,

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    • Dr. Soh received his PhD from Brown University, Providence, Rhode Island. He is a professor at the Biomedical Physics Laboratory, School of Physics, Seoul National University, South Korea. His current research interests are scientific investigations into the mechanism of acupuncture and the physical basis of acupuncture points and meridians. Dr. Lee is at the same institute. He has worked on magnetobiology and is currently studying the Bonghan system. Ms. Yoo and Ms. Baik are graduate students. Dr. Kim is at the National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University. His interest is the application of cryo-SEM for studies in microscopic anatomy.

  • Jung Sun Yoo,

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    • Dr. Soh received his PhD from Brown University, Providence, Rhode Island. He is a professor at the Biomedical Physics Laboratory, School of Physics, Seoul National University, South Korea. His current research interests are scientific investigations into the mechanism of acupuncture and the physical basis of acupuncture points and meridians. Dr. Lee is at the same institute. He has worked on magnetobiology and is currently studying the Bonghan system. Ms. Yoo and Ms. Baik are graduate students. Dr. Kim is at the National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University. His interest is the application of cryo-SEM for studies in microscopic anatomy.

  • Ku Youn Baik,

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    • Dr. Soh received his PhD from Brown University, Providence, Rhode Island. He is a professor at the Biomedical Physics Laboratory, School of Physics, Seoul National University, South Korea. His current research interests are scientific investigations into the mechanism of acupuncture and the physical basis of acupuncture points and meridians. Dr. Lee is at the same institute. He has worked on magnetobiology and is currently studying the Bonghan system. Ms. Yoo and Ms. Baik are graduate students. Dr. Kim is at the National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University. His interest is the application of cryo-SEM for studies in microscopic anatomy.

  • Ki Woo Kim,

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    • Dr. Soh received his PhD from Brown University, Providence, Rhode Island. He is a professor at the Biomedical Physics Laboratory, School of Physics, Seoul National University, South Korea. His current research interests are scientific investigations into the mechanism of acupuncture and the physical basis of acupuncture points and meridians. Dr. Lee is at the same institute. He has worked on magnetobiology and is currently studying the Bonghan system. Ms. Yoo and Ms. Baik are graduate students. Dr. Kim is at the National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University. His interest is the application of cryo-SEM for studies in microscopic anatomy.

  • Kwang-Sup Soh

    Corresponding author
    • Biomedical Physics Laboratory, School of Physics, Seoul National University, Seoul, 151-747, South Korea
    Search for more papers by this author
    • Dr. Soh received his PhD from Brown University, Providence, Rhode Island. He is a professor at the Biomedical Physics Laboratory, School of Physics, Seoul National University, South Korea. His current research interests are scientific investigations into the mechanism of acupuncture and the physical basis of acupuncture points and meridians. Dr. Lee is at the same institute. He has worked on magnetobiology and is currently studying the Bonghan system. Ms. Yoo and Ms. Baik are graduate students. Dr. Kim is at the National Instrumentation Center for Environmental Management, College of Agriculture and Life Sciences, Seoul National University. His interest is the application of cryo-SEM for studies in microscopic anatomy.

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Abstract

A staining method has been developed for in situ and in vivo observation of a threadlike tissue afloat inside the lymphatic vessels of rabbits without adherence to the vessel wall. The existence of this novel structure was not noticed previously because it is extremely difficult to detect it by microscopic inspection of lymphatic vessels. We have found a method that utilizes Janus Green B (JGB), which stained heavily the novel structure. The tissue was studied by confocal laser scanning microscopy (CLSM), light microscopy, and cryoscanning electron microscopy (cryo-SEM). The CLSM image obtained by acridine orange staining of the novel tissue revealed its characteristic nuclei distribution: rod-shaped nuclei of 10–20 μm length aligned in a broken-line/striped fashion. Hematoxylin and eosin staining revealed the threadlike structure passing through a lymphatic valve as histologically distinct from lymphatic vessels and valves. The cryo-SEM image showed the threadlike structure inside a collapsed lymphatic vessel. There were spherical globular structures observable inside sinuses in a rapidly frozen sample, which suggests liquid flowing through the longitudinal ductules in the threadlike structure. The specific staining of the JGB suggests that these threadlike structures inside lymphatic vessels have a high density of mitochondria in their cells and/or nerve-like properties, either of which may provide important clues to their physiological function. Anat Rec (Part B: New Anat) 286B:1–7, 2005. © 2005 Wiley-Liss, Inc.

INTRODUCTION

We recently reported the finding of hitherto unknown novel threadlike structures on the surfaces of internal organs (Lee BC et al., 2004b; Shin et al., 2005) as a sequel to previous reports on the threadlike structure inside blood vessels (Jiang et al., 2002; Cho et al., 2004; Lee BC et al., 2004a). These threadlike structures, first found by Bonghan Kim (1963), are thought to be subsystems of a new circulatory system entirely different from the vascular, nervous, and lymphatic systems. Kim discovered them as he sought the anatomical basis of acupuncture meridians in humans and animals. Unfortunately, his work has been neglected for a long time, mainly because Kim's discovery could not be reproduced. The existence of intravascular Bonghan duct inside the blood vessels of rats and rabbits was recently confirmed (Jiang et al., 2002; Lee BC et al., 2004a), which was followed by the observation of similar structures on the surfaces of internal organs (Shin et al., 2005).

One of the objections raised by medical or veterinarian experts to the existence of the Bonghan ducts was the possibility of confusion with lymphatic vessels (Kellner, 1966; Kroger, 1973). Indeed, both objects are semitransparent, have similar sizes, and carry liquid. Thus, it is critical to provide clear evidence to distinguish them. In the case of the threadlike structures on the surfaces of internal organs, Feulgen reaction study provided the method to allow the visualization of features that distinguish the novel structure from a lymphatic vessel (Shin et al., 2005). The purpose of the present article is to demonstrate that the Bonghan duct is completely different from a lymphatic vessel. We provide visual evidence of Bonghan ducts inside a lymphatic vessel that did not adhere to the vessel wall, but passed through lymphatic valves. This was achieved by a staining method using Janus Green B (JGB).

Since the discovery of the lymphatic vessel, its anatomical structure and physiological function have been important subjects of many investigations (Podgrabinska et al., 2002; Hangai-Hoger et al., 2004). It is well established that there is present a flow of liquid, which includes lymphocytes in the lumen of the lymphatic vessel, and no other structure inside the lymphatic vessel is known. Thus, it is rather surprising that the threadlike structure (Bonghan duct) afloat inside lymphatic vessels has not been noticed at all despite many studies and surgeries on the lymphatic vessel. However, it is not uncommon in biological science that transparent tissues cannot be detected without using appropriate staining techniques.

In this article, we report the JGB staining method that reveals an almost transparent threadlike structure inside the lymphatic vessels of rabbits in situ and in vivo. JGB is known as a dye capable of staining mitochondria and nerves (Cooperstein et al., 1960; Yack, 1993) and in this study makes the threadlike structures clearly visible. Several other dyes did not exhibit any such visualization capacity. In order to prove that the JGB-stained threadlike structure inside lymphatic vessels was not an optical illusion or in any way a visual artifact but rather real tissue, a piece of the threadlike sample was taken and observed using confocal laser scanning microscopy (CLSM) after staining by 0.1% (w/v) acridine orange, an agent that characterizes nuclei and their distribution. Furthermore, in order to demonstrate that there is a novel structure inside the lymphatic vessel, the lymphatic vessel with the threadlike structure was studied using a histological method and cryoscanning electron microscopy (cryo-SEM). Cross- and oblique sectional images of the tissue show its peculiar structure, and a cryo-SEM image shows a detailed internal architecture of the structure, which was maintained by a rapid freezing of the samples.

BONGHAN DUCT STAINING WITH JGB

Animals and Sample Preparation

New Zealand white rabbits (14 females; 12 weeks old; mean weight, 2.1 kg) were obtained from Korean Jung-Ang Laboratory Animal for use in this study. All of the animals had ad libitum access to food and water. The procedures and the care of the animals conformed to current international laws and policies (Guide for the Care and Use of Laboratory Animals, 1996). The rabbits were anesthetized with urethane (1.5 g/kg) administered intraperitoneally and all surgical procedures were performed under general anesthesia.

The left lateral sides or middle lines of the abdomens of the rabbits were incised, and the large lymphatic vessels beside the inferior vena cava were exposed. In order to stain a threadlike structure afloat inside a lymphatic vessel, approximately 0.3 ml of JGB [dye content 65% Janus Green B (Aldrich) 1 g in 99.9% ethyl alcohol 100 ml] preheated at 40°C was injected as slowly as possible into a lymph node or lymphatic vessel close to the inferior vena cava using a fine needle of gauge 30.5. After the injection needle was carefully withdrawn, the blue color of JGB flowing inside the lymphatic vessel was observed. Then the preheated phosphate-buffered saline (pH 7.4) at 40°C was injected into the lymphatic vessel and lymph node in order to wash out the backward-flowing JGB and to keep them warm and maintain their vital condition. Careful observation was carried out under a stereomicroscope. The position of well-stained threadlike structure in the lymphatic vessel varied according to the staining and lymphatic vessel conditions of the subject rabbits. When the well-stained part was found, the careful removing of connective tissues around the lymphatic vessel was carried out to allow clear observation and sample removal. The specimens were fixed in 10% neutral buffered formalin as quickly as possible, and then part of them were stained by 0.1% (w/v) acridine orange (Sigma-Aldrich) in PBS for CLSM (LSM510; Carl Zeiss).

Histology

For histological study, the lymphatic vessels with threadlike structures were fixed in 10% neutral buffered formalin for paraffin embedding. Sections of 5 μm thickness were cut with a microtome and stained with hematoxylin and eosin. The sections were observed and photographed under a light microscope (Axiophot; Carl Zeiss). For cryo-SEM observation, the sample fixed by the above method was packed into a rivet that was mounted onto a slotted metal stub and plunged into liquid nitrogen for cryofixation for 20 sec. Frozen specimens were placed in a cryochamber of a cryotransfer system (CT1500; Oxford Instruments, Oxon, U.K.) maintained at −170°C and transferred to the cold specimen stage of a scanning electron microscope (JSM-5410LV; JEOL, Tokyo, Japan). The specimen was then freeze-fractured to expose the internal structure of frozen lymphatic vessel containing JGB-stained threadlike structure that protruded above the rivet surface in the cryochamber using a cold knife. Sublimation of superficial frozen water was performed by heating and maintaining the cold specimen stage at −60°C for 10 min and the process was monitored with the electron microscope at an accelerating voltage of 5 kV. The specimens were sputter-coated with gold (approximately 30 nm in thickness) in the cryochamber and observed with an electron microscope at 20 kV.

BONGHAN DUCTS WITHIN LYMPHATIC VESSELS

A stereomicroscopic view of the lymphatic vessel around the inferior vena cava is shown in Figure 1A, where the diameter is approximately 600 μm. Notice that blood capillaries on the wall of the lymphatic vessel are clearly visible and even those below the bottom wall of the lymphatic vessel can be seen. One can also notice the lymphatic valve, although not so clearly. However, there was not even a slight hint of any other structure inside the lymphatic vessel. This is a typical situation in that the novel tissue had eluded observation with an ordinary microscope. An unexpected structure inside the lymphatic vessel was visualized after the staining dye JGB was injected into either the lymphatic vessel or lymph node. Figure 1B and C are stereoscopic images after the injection of JGB into lymphatic vessel or lymphatic node. The JGB-stained threadlike structure was clearly observed, with a stained lymphatic valve and weakly stained lymphatic vessel wall, as seen in Figure 1B. Another very thick threadlike structure was observed in a cylindrical form between two lymphatic valves, as in Figure 1C. The thickness of the threadlike structures varied widely from one subject to another, as illustrated in Figure 1B and C. The inset in Figure 1C shows that a JGB-stained threadlike structure is floating through three stained lymphatic valves inside the weakly stained lymphatic vessel wall. JGB helped to visualize the threadlike structure more effectively than other dyes employed, i.e., methylene blue, methyl green, and toluidine blue. Those dyes stained the lymphatic vessel as heavily as the threadlike structures, reducing or even obliterating the value of the staining procedure.

Figure 1.

A stereoscopic image of lymphatic vessel around the inferior vena cava of a rabbit. Typical view without staining (A) and after injection of JGB into the lymphatic vessel (B and C). In A, lymphatic valves (arrowhead) inside the lymphatic vessel and blood capillaries around the lymphatic vessel wall are observable. After JGB injection, the threadlike structure (arrow) in B is clearly exhibited with a stained lymphatic valve (arrowhead) inside the weakly stained lymphatic vessel wall. The threadlike structure (arrow) in C is the biggest one among those observed, whose size is approximately 600 μm in diameter. In the inset, three stained lymphatic valves (arrowheads) indicate the size of the lymphatic vessel and the well-stained novel structure may be seen lying in and through those valves. The blue color of JGB in B is deeper than in C due to higher concentration of the dye. Scale bars = 1 mm.

Figure 1B and C are typical of the images obtained from the 14 experiments presented in Table 1. We examined the abdominal lymphatic vessels connected to a lumbar node beside the inferior vena cava and iliac veins and the lymphatic vessels in the peritonea around the intestines. During the 14 experiments, we observed the intravascular structure of the lymphatic vessels from every rabbit except for two. The JGB-stained threadlike structure was therefore not an abnormal state of one particular subject. The diameters of the lymphatic vessels we examined ranged from 130 to 2,000 μm and the diameters of the threadlike tissues ranged from 26 to 500 μm, with an average of 129 μm. The thickness of the threadlike structures varied widely from one subject to another, which was also noticed in the similar structures on the surfaces of mammalian organs (Shin et al., 2005).

Table 1. Size data of nineteen threadlike structures from rabbits (14 females, 12 weeks old)
SubjectInjection SiteLymph Vessel Position Where Sample is TakenThe Diameter of the Lymph Vessel (μm)The Diameter of the Threadlike Structure (μm)
NumberWeight (kg)
  1. IVC, around the inferior vena cava; LI, on a large intestine; IV, around the iliac vein; M (m), maximum (minimum) diameter of lymphatic vessel; DM (Dm), maximum (minimum) diameter of the threadlike structure; X, failure to obtain the sample.

11.8Lymph vessel along caudal vena cavaIVCM: 1500100
    m: 600 
22.2Lumbar nodeIVC200050
32.0Lumbar nodeIVC106080
42.2Lumbar nodeIVC10016
    880DM: 510
     Dm: 190
52.2Lymph vessel along caudal vena cavaIVCM: 40030
    m: 180 
62.2Lumbar nodeIVC54080
72.5Lumbar nodeIVCXX
82.3Lumbar nodeIVCXX
91.8Lumbar nodeIVC18050
    600230
101.8Lumbar nodeIVC1500500
    1300DM: 400
     Dm: 170
    960170
112.2Lymph vessel on the large intestineLI36026
121.8Lumbar nodeIVC13040
    540260
132.2Caudal lymph nodeIV27044
    36050
142.2Caudal lymph nodeIV90036
Number of subjectsAverage weightAverage ± Standard DeviationIVC786 ± 538154 ± 143
142.1    
   LI36026
   IV510 ± 34043 ± 17

Confocal laser scanning microscope (LSM510; Carl Zeiss) observation elucidated the distribution of the nuclei of the threadlike structures (Fig. 2). One part of the structure of the threadlike structure was magnified 400 times and optically sectioned by 1 μm. This sectioned image clearly revealed that the threadlike structure contained characteristic rod-shaped nuclei whose lengths are approximately 10–20 μm, and that these nuclei are arranged in a broken-line striped fashion. The numbers of broken-line stripes vary in the four panels of Figure 2, in which the focal points have been moved by 1 μm one by one from the bottom. These features imply that the threadlike structure could consist of a bundle structure consisting of small subduct the diameter of which is approximately 5 μm. This structure is also consistent with previous reports on similar structures inside blood vessels of rats and swine (Cho et al., 2004; Lee BC et al., 2004a) and on the internal organ surface of rabbits and rats (Lee BC et al., 2004b; Lee KJ et al., 2004; Shin et al., 2005)

Figure 2.

Optically sectioned serial images of threadlike structure after acridine orange staining were taken by CLSM. The nuclei are rod-shaped and 10–20 μm long. They form a broken-line array, and the number of lines varies from left panels to right, in which the focal depth is increased by 1 μm from the bottom of the threadlike structure. Scale bars = 10 μm.

A sectional image was taken after staining hematoxylin and eosin to get information on the location of the novel structure inside the lymphatic vessel and on its cells and sinuses. A cross-sectional image shows clearly that there is a distinctive tissue element not attached to the lymphatic vessel wall (see insert of Fig. 3A). Figure 3A shows that the novel tissue has an outer membrane and contains heavily stained nuclei distinctively different from the endothelial cells in the lymphatic vessel. There were several big and small sinuses in the extracellular matrix inside this novel tissue. These sinuses appeared continuously at the same site in several separate slides. The continual appearances of sinuses slide by slide suggest that they are subducts that require further study along with efforts to elucidate the physiological function of this new tissue. The oblique section in Figure 3B shows a remarkable image of the threadlike structure mostly surrounded by a lymphatic valve. This image and the inset depict the passage of the threadlike structure through the valve. This striking picture is an indisputable demonstration of the real existence of the threadlike structure.

Figure 3.

A: Cross-sectional image of the threadlike structure (arrow) inside a lymphatic vessel stained by hemtoxylin and eosin (1,000×) with a wide-view insert (200×). B: Oblique sectional image (400×) of the threadlike structure (arrows) with a wide-view inset (200×). As seen in the insets, the novel tissue did not adhere to the lymphatic vessel wall and lymphatic valve (arrowheads). There are several big and small sinuses (asterisks) partially surrounded by heavily hematoxylin-stained nuclei (dashed arrow) and these consistently appear at the same site along the threadlike structure. Scale bars = 20 μm; 50 μm for inserts.

This novel tissue has characteristic features present in the nuclei as well as the many sinus-like structures in extracellular matrix. Beyond the reach of a morphological method like CLSM or the histological work with hematoxylin and eosin staining, cryo-SEM enables one to interpret the presence of liquid in samples as well as cellular structures (Read, 1991). Soluble materials, organelles, and macromolecules are retained in segregation zones during freezing. This method also revealed the detailed morphology of the threadlike structures inside the lymphatic vessel.

In the freeze-fractured specimen, the lymphatic vessel surrounded by connective tissue was found to possess an undulated outer membrane system after freezing followed by sublimation, but the novel tissue was considered to be inside the collapsed lumen of the lymphatic vessel (Fig. 4A). This novel tissue appeared to have a distinct outer membrane that did not collapse, and the dimension of this particular specimen was similar to that found in the stereoscopic image Figure 1C. As shown in the rectangle of Figure 4A and its magnified view in Figure 4B, the sinus in this novel tissue has a wider spacing of segregation zones than the surrounding cytoplasm (Fig. 4B). Even the sinus has segregation zones and a spherical globular structure, which implies that there was a liquid-containing spherical body and other components inside the sinus.

Figure 4.

A: The freeze-fractured specimen shows that the collapsed lymphatic vessel (outlined with a dashed line) contains another substructure, which has a distinctive outer membrane. B: Higher magnification of the rectangle in A. Note that the novel structure had sinuses with segregation zones (arrowheads) and a ball-like globular structure (arrow). In the sinus, fluid material (arrowheads) flowed with the globular structure. Scale bars = 200 μm (A); 20 μm (B).

DISCUSSION

The search for the intravascular threadlike structure floating inside a lymphatic vessel undertaken in this study was motivated by previous work in which these threadlike structures were found inside the blood vessels of rabbits and rats (Lee BC et al., 2004a). As mentioned earlier, the breakthrough reported here is the discovery that the dye JGB can strongly stain the novel intravascular threadlike tissues, which makes it possible to observe them in situ and in vivo inside the transparent lymphatic vessels. This, in turn, convincingly demonstrates the evident existence of the novel structures. In the earlier work on the intravascular structure of blood vessels, we had been forced to use complicated processes such as a special perfusion method, an acridine orange fluorescence technique, and hematoxylin and eosin staining to prove the existence of these novel structures (Lee BC et al., 2004a, 2004b). Hence, the discovery of the JGB staining technique that enabled the in situ and in vivo demonstration method of the novel structure inside lymphatic vessels was crucial for establishing its existence. This method was almost simultaneously developed with a magnetic nanoparticle technique by certain of the current authors (Johng et al.). These two approaches use different materials and methods, but their results are in good agreement. To the best of our knowledge, the photographic images of the sections of the lymphatic vessels with the threadlike structures, as well as the stereoscopic views, were obtained for the first time in these two studies.

In the early 1960s, Bonghan Kim reported finding an intravascular threadlike structure inside lymphatic vessels as a part of a large network of a new circulatory system entirely distinct from the vascular or neural systems (Kim, 1963). Unfortunately, he kept his method secret, so no one was able to reproduce his results. His work has therefore been neglected, as one might expect, for a long time. In an exception to this neglect, the Japanese anatomist Fujiwara (1967) was in fact able to confirm Kim's results partially, but his work has not attracted much attention either. Only very recently have investigators rediscovered the intravascular threadlike structure using a slow-perfusion method in rats and rabbits (Cho et al., 2004; Lee BC et al., 2004a) as well as threadlike structures on the internal organ surfaces of rabbits and rats (Lee BC et al., 2004b; Lee KJ et al., 2004; Shin et al., 2005). Even though Kim claimed the existence of the threadlike structure inside lymphatic vessels, he did not present any photographic evidence. As far as we know, no one has been able to confirm his claim on the lymphatic Bonghan ducts.

JGB has been known to stain mitochondria and nerves (Cooperstein et al., 1960; Yack, 1993). According to Kim's description, the threadlike structures were said to have unique patterns of movement, different from gastrointestinal ducts, and to be parasymphathomimetic (Kim, 1963). This suggests that the Bonghan ducts are stained by JGB because they have a large number of mitochondria, allowing for unique peristaltic movements, and/or have nerve-like properties. This characterization would have deep physiological implications and will need to be elucidated in the future. The revelation of a high density of mitochondria in the threadlike structure was suggested for the first time in this study.

The hematoxylin and eosin-stained sectional images, and the freeze-fractured sample images obtained by cryo-SEM, show convincingly that certain threadlike structures exist floating inside the lymphatic vessel. The thickness of the threadlike structures varies widely from one subject to another. The thickness may depend on the functional state of the threadlike structures and/or the physiological state of the subject animal. The diameter and bundle structure, and the size and distribution of endothelial nuclei of the intravascular threadlike structures in various lymphatic vessels, were similar to those found in arteries and veins of rats and swine (Cho et al., 2004; Lee BC et al., 2004a). The bundle structure of the threadlike structure composed of subducts, the size of which is approximately 5 μm, is inferred from the CLSM and histological images and this feature is consistent with that of the threadlike structures on the mammalian organ surfaces (Shin et al., 2005). Furthermore, the noticeably spherical globular structures and segregation zones observable in the cryo-SEM image imply that there was liquid inside the sinuses of the subducts.

The current study is the first work to provide a stereomicroscopic view of novel JGB-stained threadlike structures as well as light microscopic cross-sectional images that vividly exhibit the passage of these structures through lymphatic valves inside lymphatic vessels. The discovery of the dye that strongly stains the novel threadlike tissue and reveals its histological structure is a critical contribution toward an important extension of the current understanding of cellular anatomy.

With these results, it is reasonable to consider more seriously Kim's statements about the putative physiological function of this structure (Kim, 1965). His work was intended to establish the physical bases for acupuncture meridians and acupoints. Recently, an anatomical investigation (Langevin and Yandow, 2002) and ultrasonic imaging of acupoints have revealed structures in agreement with his claims (Jones and Bae, 2004). However, both Kim's theory and its relation to the currently widely used neurophysiological models (Cho et al., 1998) need to be further investigated. According to Kim (1965), some sort of liquid with granules containing DNA (Shin et al., 2005) flows through the ducts, which are similar to microcells and might be somehow related to “cell therapy.” In addition, the threadlike ducts were considered to be involved with developmental processes and, in particular, the lymphatic intravascular threadlike structure was related to immunological and hematopoietic function (Kim, 1965). Whatever the eventual outcome of deeper investigation of these claims, the finding of the novel structure inside lymphatic vessels is not a mere curiosity but rather a herald of a breakthrough in establishing the third circulatory system that consists of the Bonghan ducts inside blood vessels, on the organ surfaces, and under the skin. Further studies of its histological aspects and physiological functions suggest the possibility of new insights in both biology and medicine as well as acupuncture theory.

Acknowledgements

The authors are grateful to Ms. Eun-Jung Kang in the National Center for Inter-University Research Facilities for the use of confocal laser scanning microscope, to Ms. Young Soon Kim in the National Instrumentation Center for Environmental Management of Seoul National University for cryoscanning electron microscopic photography, and to Ms. Eun-Sook Cho in the College of Medicine Seoul National University for hematoxyline and eosin staining. This work is supported in part by the Ministry of Science and Technology (NRL, M1-0302-00-0007).

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