BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a visualizing agent comprising a magnetic nanoparticle, and a visualizing method by using the same. More specifically, the method comprising A method of visualizing the intravascular threadlike structure floating inside a lymphatic vessel comprising injecting a magnetic nanoparticle into the lymphatic tissue to be stained, and conducting microscopy.

Description of the Related Art

In the early 1960's, Bonghan Kim reported finding an intravascular threadlike structure inside lymphatic vessels as part of a large network of a new circulatory system entirely distinct from the vascular or neural systems (1), but 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. Bonghan Kim, a North Korean physiologist, reported his findings on the physical bases of acupuncture meridians, which were named "Bonghan ducts" and supposedly formed a third circulatory system. Through the network of Bonghan ducts flows a liquid that contains special granules that have the power to regenerate cells in damaged tissues. His work was published in five articles from 1962 to 1965 in North Korean medical journals (in Korean). The 1st reference (1) is the only English monograph that has summarized the earlier part of his work.

In an exception to this neglect, the Japanese anatomist Fujiwara was in fact able to partially confirm Kim's results (2, 3) but his work has not attracted much attention either. Only very recently have certain investigators rediscovered the intravascular threadlike structure using a slow perfusion method in rats and rabbits (4, 5) as well as threadlike structures on the internal organ surfaces of rabbits and rats (6, 7, 8). Even though Kim claimed the existence of the threadlike structure inside lymphatic vessels, he did not present any photographic evidence. As far as no one has been able to confirm his claim, including Fujiwara, mainly because Kim's method had not been disclosed.

Since quite early after the discovery of the lymphatic vessel, its anatomical structure and physiological function have been thought to be mostly, if not completely, understood (9). It is firmly 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.

The existence of this novel structure was not noticed previously because it is extremely difficult to detect it by microscopic inspection of lymphatic vessels. In Western studies of anatomy, there is not even the slightest mention of intravascular threadlike structures afloat inside lymphatic vessels (10). The fact that such structures exist on a meso-scale (with thicknesses on the order of 20 μm) and have not been noticed at all in numerous surgical operations is quite a surprise. Undoubtedly, their optical transparency makes them extremely difficult to detect.

SUMMARY OF THE INVENTION

To resolve the problem that the intravascular threadlike structure floating inside a

lymphatic vessel has not been visualized, the inventors of the present invention have found a novel method which utilizes magnetic nanoparticles which stained heavily the novel structure so that direct manifestation was achieved. Thus it is surprising to discover a threadlike structure afloat inside lymphatic vessels that does not adhere to the vessel wall. It rightly sounds extremely unlikely that threadlike structures of a diameter of about 20 μm could have gone unnoticed despite the many surgeries and studies on the lymphatic vessels throughout the world.

Thus, the object of the present invention is to provide a visualizing agent comprising a magnetic nanoparticle labeled organic dye to vividly exhibit the passage of these structures through lymphatic valves inside lymphatic vessels under stereomicroscope. The discoveries of the dye that strongly stains the novel threadlike tissue and reveals its histological structure are critical contributions toward an important extension of the current understanding of cellular anatomy.

Another object of the present invention is to provide a method of visualizing a Bonghan Duct inside a lymphatic vessel which comprises injecting a magnetic nanoparticle labeled with a luminescent organic dye into the lymphatic tissue, allowing the uptake of the magnetic nanoparticle in the tissue, and performing microscopy observation.

The still object of the present invention is to provide a visualizing method, wherein the magnetic nanoparticle stains the Bonghan duct in situ or in vivo. The object of the present invention is to provide a visualizing method, wherein a magnetic field is applied to the lymphatic tissue after the injection of the nanoparticle in the lymphatic tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a stereoscopic image of lymphatic vessel around the inferior vena cava of a rat in situ in accordance with Example 1;

Fig. 2(A) is microscopic images of a differential interference contrast image and Fig. 2(B) is a fluorescence image of the same specimen, and Fig. 2(C) is a result of acridine orange staining showing the distribution of nuclei in this threadlike structure in accordance with Example 1 ;

Fig. 3 is confocal laser scanning microscope images of cryo-sectioned threadlike and corpuscular structures; Fig. 4 is confocal laser scanning microscope images of cryo-fϊxed threadlike and corpuscular structures: (A) differential interference contrasts, (B) nanoparticle-fluorescent images, (C) magnified views of the corpuscles;

Fig. 5(A) is an anatomical illustration of the lymph nodes and vessels which was located near the caudal vena cava, and Fig. 5(B) is the Bonghan duct and the corpuscle inside the lymphatic vessel in situ and in vivo; and

Fig. 6 A is a hematoxylin-eosin staining to perform histological studies of the novel threadlike and corpuscular structures in accordance with Example 2.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS The present invention is described in more detailed hereinafter. The accompanying drawing, which is included to provide further understanding of the invention and is incorporated in and constitutes a part of this specification, illustrates an embodiment of the invention and together with the description serves to explain the principles of the invention.

A visualizing agent used for staining the Bonghan duct inside a lymphatic vessel comprising a magnetic nanoparticle labeled with a luminescent organic dye. The agent is used for visualizing the Bonghan duct and/or Bonghan corpuscle in situ and in vivo. The agent visualizes the Bonghan duct and/or Bonghan corpuscle by heavier staining than other lymphatic tissue.

In addition, the present invention provides a method of visualizing a Bonghan Duct inside a lymphatic vessel which comprises injecting a magnetic nanoparticle labeled with a luminescent organic dye into the lymphatic tissue, allowing the uptake of the magnetic nanoparticle in the tissue, and performing microscopy observation. For the first time novel threadlike structures were visually demonstrated in vivo inside the lymphatic vessels of rats by injecting fluorescent magnetic nanoparticles into lymph nodes. These hitherto unobserved structures disclosed themselves by taking up nanoparticles more heavily than the lymphatic vessels. Due to the fluorescence of the nanoparticles, microscope image of specimens of lymphatic vessels vividly exhibited the threadlike structures and their associated corpuscles. These threadlike structures are thought to be a part of another circulatory system different from the blood vascular and the lymphatic systems; this radical challenge to current anatomy has yet to be completed.

Hereinafter, the intravascular threadlike structure floating inside a lymphatic vessel is so called as "Bonghan duct" which Bonghan Kim reported as part of a large network of a new circulatory system entirely distinct from the vascular or neural systems. According to

Bonghan Kim's description, the threadlike structures were said to have unique patterns of movement, different from gastrointestinal ducts, and to be parasymphatomimetic.

In the present invention, the magnetic nanoparticle has the structure including a

core of cobalt-ferrite particle and a shell of amorphous silica coated on the core, and the surface of the magnetic nanoparticle is modified by the biocompatible polymer. The organic dye is rhodamine B isothiocyanate, or fluorescein isothicyanate. The example of the flurorescein isothicyanate includes terra-methyl rhodamine B isothiocyanate (RITC, emission wave length ^ _ax =SSS nm), but is not limited thereto. The biocompatible polymer is particularly limited, and the example includes PEQ but is not limited thereto. An important factor contributing to the threadlike structures' visibility in vivo was the nanoparticles' being more strongly taken up by them than by the lymphatic vessel. The average particle size of the magnetic nanoparticle can be lnm to lOOnm. In an embodiment of the present invention, Cobalt-ferrite magnetic nanoparticles with an average size of about 9 nm were coated with a shell of amorphous silica containing the luminescent organic terra-methyl rhodamine B isothiocyanate (RITC, emission wavelength X _m2x =SSS nm). The surface of core-shell magnetic nanoparticles was further modified with a biocompatible poly (ethylene glycol) (PEG) oligomers. The total size of the core-shell structure was about 50 nm. The volume of dye injected was 0.01 ml. The concentration of nanoparticles (MNP-SiO ₂ (RITC)-PEG) was 2.0 mg/cc, and they were suspended in sterile saline solution at pH 7.4. For more details, see T. J. Yoon et al., Angew. Chem. Int. Ed. 44, 1068 (2005) (Ref. 11).

In the preferred embodiment, the injection of fluorescent magnetic nanoparticles into two lumbar lymph nodes and the application of a magnetic field above the nodes and the lymphatic vessels connected to them. The application of the magnetic field is a critical process without which the nanoparticles will flow away with lymphocytes rather than being taken up by the cells in the threadlike structures. That is, the threadlike structure would not

have absorbed a sufficient amount of nanoparticles to allow the structure to be detected without the magnetic field application. The magnetic field is applied to the lymphatic tissue in the magnetic field strength of O.lto 0.5 Tesla at surface, for 1 to 120 minute, more preferably 10 to 40 minutes. In a specific embodiment, after injection of the magnetic nanoparticles, a disk-type commercial Nd-Fe-B magnet (diameter = 14 mm, thickness = 5 mm, magnetic field strength = 4000 gauss at the surface) was put on the lymphatic vessels connected to the lumbar nodes. The role of the magnet was to hold the nanoparticles at the node and the lymphatic vessel so that they had enough time to be taken up by the cells in the threadlike structure. This process was important for detecting the threadlike structure. Without this process, the nanoparticles would have flowed with the lymphatic flow, and the threadlike structure would not have absorbed a sufficient amount of nanoparticles to allow the structure to be detected. The magnetic field was applied for 20 minutes.

In an embodiment of the present invention, the lymphatic tissue such as the lymph nodes and vessel are located near the caudal vena cava. An anatomical illustration is shown in Fig. 5A. The nanoparticles are injected into the two lumbar nodes. The magnetic field is applied to the region indicated by the red circle. The observed lymphatic vessels are inside the blue rectangle. Fig.5B shows, in situ and in vivo, the threadlike structure and corpuscle inside the lymphatic vessel. For clear observation, it is necessary to produce as much contrast as possible. A piece of black paper was put under the lymphatic vessel in order to isolate the vessel from its complicated surroundings so that the lymphatic vessel alone could be examined. This turned out to be a very simple, but effective, way for enhancing the contrast. Two light

sources, halogen and mercury lamps were used, and optical fibers with diameters of 3 mm to illuminate the target area. The illuminating direction was about 10° from the horizontal plane (polar angle = 80°), and the azimuthal angle was about 45° from the lymphatic vessel line. Fine tuning the direction of illumination was important for detecting the barely visible threadlike structures, so the direction was adjusted by pressing the optical fiber, which was held by a micro-manipulator, slightly. Once the target structure had been detected, the angle was adjusted by fine tuning to achieve the best contrast.

The stained intravascular threadlike structure floating inside a lymphatic vessel is observed with a microscopy. The microscopy is not particularly limited, and microscope being capable of imaging the stained structure used in conventional art may be used in the present invention. For examples, the microscopy observation can be performed with preferably a stereomicroscope, a light microscope, an inverted microscope, a confocal laser scanning microscope (CLSM), or a cryo scanning electron microscope (Cryo-SEM).

In an embodiment of the present invention, the visualizing method further includes an acridine orange staining of the Bonghan duct after allowing the uptake of the magnetic nanoparticle. Then, the microscopy observation is performed with a confocal laser scanning microscope, or an inverted microscope. The inverted microscope can be performed in a mode of a differential interference contrast, or a mode of red or green fluorescence.

In an embodiment, the visualizing method further includes hematoxylin and eosin staining of the Bonghan duct, and then performing the microscopy observation with a light microscope, for histological study.

The present invention is described in more detailed hereinafter by using the drawings.

Fig. IA shows a typical stereomicroscope view of a lymphatic vessel near the caudal vena cava of a rat. The lymphatic vessel (outlined with a dashed line) appears transparent to the extent that the lymphatic valve (double line arrows) and even the blood capillaries (arrow heads) in the fats under the lower vessel wall can be seen. The inside of the lymphatic vessel is so clearly exposed to view that one can hardly imagine the existence there of anything other than the transparent lymphatic fluid. However, simple observations can be misleading, as is demonstrated by Fig. IB, which reveals the threadlike structure (thick arrow) in vivo and in situ. The structure is visible due to the fluorescence of the nanoparticles injected into the lymph nodes. Corpuscles (arrows), which are sparsely located along the lymphatic vessel and which are connected by the threadlike structures, as well as valves, can also be seen. A piece of black paper was put under the lymphatic vessel for clarity and ease of observation. A more magnified view of a section of the lymphatic vessel in Fig. IB that includes a valve (double line arrow) and a corpuscle (arrow) is shown in Fig. 1C. A piece of a lymphatic vessel similar to the specimen depicted in Fig. IB was taken, fixed with neutral buffered formalin (NBF 10%), put on a slide, and examined under an inverted microscope. The differential interference contrast image in Fig. 2A shows the threadlike structure (thick arrow) made partially protrude from the lymphatic vessel (dotted arrow). One end of that vessel had been torn off using a sharp micro-needle, but the debris had not been removed. A fluorescence image of the same specimen is shown in Fig. 2B. Even after having been fixed with NBF, the threadlike structure is very bright clearly proving its existence, due to the fluorescence of the uptaken nanoparticles. The distribution of nuclei (arrow heads) in this threadlike structure was revealed by acridine orange staining

(Fig. 2C).

A confocal laser scanning microscope image of a cross section of the novel threadlike structure (NTS) is shown in Fig. 3A. The NTS (thick arrow) is brighter than the vessel wall (dotted arrow) because it absorbs more nanoparticles than the wall does. In a less sensitive image (Fig. 3B), the lymphatic vessel is no longer visible, and there appear dark holes (arrow heads) in the NTS. The dark holes are the locations of nuclei. The nanoparticles were taken up by the cytoplasm but not by the nuclei (11). The double image of acridine orange and the nanoparticle shows nuclei by green color at the dark holes and in the vessel wall (dotted arrow) (Fig. 3C). The specimen was prepared in a cryo-section with a thickness of about 6 μm.

Confocal laser scanning microscope images of cryo-sectioned threadlike and corpuscular structures inside a lymphatic vessel with a valve are shown in Fig. 4. The differential interference contrast image and the fluorescence image of the nanoparticles are shown in Figs 4A and 4B, respectively. The corpuscle (arrow) is to the right in the lymphatic duct (dotted arrows), and the entwined valve (double line arrows) is in the middle. To the left side, a threadlike structure (thick arrow) can be seen. Fig. 4B shows that the threadlike structure and the corpuscle are far brighter than the lymphatic valve and the lymphatic walls, which implies that the nanoparticles are taken up more strongly by the novel structures as previously mentioned. Two large holes with spherical bodies inside are clearly seen in Fig. 4(B). In the magnified views of the corpuscles in Fig. 4C, two large holes with spherical bodies inside are clearly seen. These are thought to be sinuses through which some liquid and granules flow, and to be deeply involved in the physiological functions of the novel structure.

The physiological function of the Bonghan ducts and corpuscles is related to developmental and hematopoietic processes according to Kim (12) and has yet to be studied. Some sort of liquid with granules flows through them. These granules are spherical bodies with diameters of about 1 to 2 μm and contain DNA and cytoplasm surrounded by a thin outer membrane (12), as was observed by Fujiwara (3) and more recently by two other groups (7). The inventors of the present invention used Feulgen reaction staining to demonstrate the existence of DNA in these granules (8). These granules are similar to microcells in shape, size, and DNA content; In addition, both the granules and microcells are surrounded by thin membranes (12, 13). Microcells have been widely used for cell fusion studies, and have provided a tool for investigating carcinogenesis (14) and Downs Syndrome (15). The main difference is their sources: Microcells are produced in vitro by chemical agents such as colcemid and cytochalasin B (16), and they are observed in pathological situations such as large interphase sarcoma cells (17) whereas the novel granules are thought to be naturally generated in normal tissues and to flow through a network of the threadlike structures. These granules are able to regenerate cells in the damaged tissues of an injured organ (12), which might have profound medical implications akin to contemporary cell-therapy.

The present invention will now will be described more fully hereinafter with reference to the accompanying examples, in which exemplary embodiments of the invention are shown.

EXAMPLE 1

Preparation of the nanoparticle solution: Organic dye-labeled magnetic

nanoparticles were synthesized in the laboratory of one of the inventors (J. K. Lee). Cobalt- ferrite magnetic nanoparticles with an average size of about 9 nm were coated with a shell of amorphous silica containing the luminescent organic tetra-methyl rhodamine B isothiocyanate (RITC, emission wavelength nm). The surface of core-shell magnetic nanoparticles was further modified with a biocompatible poly (ethylene glycol) (PEG) oligomers. The total size of the core-shell structure was about 50 nm. The volume of dye injected was 0.03 ml for each lymph node. The concentration of nanoparticles {MNP- SiO ₂ (RITC)-PEG) was 2.0 mg/cc, and they were suspended in sterile saline solution at pH 7.4 to obtain a magnetic nanoparticle solution. For more details, see T. J. Yoon et al., Angew. Chem. Int. Ed. 44, 1068 (2005) (Ref.11).

Animal preparation and surgical procedure: Eleven Wistar rats (10 males and one female) and two Sprague-Dawley males of about 200 grams were obtained from Jung Ang Laboratory Animal Company for use in this study. The animals were housed in a constant-temperature controlled environment (23 ⁰ C) with 60% relative humidity under a 12-h light/dark cycle. All of the animals had ad libitum access to food and water. The procedures involving the animals and their care were in full compliance with current international laws and policies (Guide for the Care and Use of Laboratory Animals, National Academy Press, 1996). The rats were anesthetized with urethane (1.5g/kg) administered intraperitoneally, and all surgical procedures were performed under general anesthesia. Under deep anesthesia the abdominal sides of the rats were incised, the lumbar nodes near the caudal vena cava were located and exposed by removing the surrounding peritonea and fats.

Illumination: For clear observation, a piece of black paper was put under the lymphatic vessel in order to isolate the vessel from its complicated surroundings so that the lymphatic vessel alone could be examined. Two light sources, halogen and mercury lamps were used, and optical fibers with diameters of 3 mm to illuminate the target area. The illuminating direction was about 10° from the horizontal plane (polar angle = 80°), and the azimuthal angle was about 45° from the lymphatic vessel line. Fine tuning the direction of illumination was important for detecting the barely visible threadlike structures, so the direction was adjusted by pressing the optical fiber, which was held by a micro-manipulator, slightly. Once the target structure had been detected, the angle was adjusted by fine tuning to achieve best contrast.

Microscopes: The surgery and the observation of the threadlike structure were done with a stereomicroscope (SZX12, Olympus) and were recorded with a CCD camera (DP70, Olympus). The light microscope used to obtain the images in Fig. 2 was an inverted microscope (Olympus 1X71), and the images in Fig. 6 were obtained with a light microscope (Olympus BX51). The confocal laser scanning microscope images were obtained with a Zeiss LSM 510 CLSM instrument.

1-1 Stain of the lymphatic tissue in rat

Into two lymphatic nodes of the thirteen rats as described above, a fluorescent magnetic nanoparticle solution (0.03 ml for each node) was slowly injected over 2-4 minutes by using a 30-gauge needle. The two nodes are shown in Fig. 5A. The nanoparticle

solution was injected into the two lumbar nodes. After injection of the magnetic nanoparticles, a disk- type commercial Nd-Fe-B magnet (diameter = 14 mm, thickness = 5 mm, magnetic field strength = 4000 gauss at the surface) was put on the lymphatic vessels (dotted arrows) connected to the lumbar nodes to apply the magnetic field for 20 minutes. The magnetic field was applied to the region indicated by the red circle in Fig. 5A. The observed lymphatic vessels were inside the blue rectangle. Fig. 5B shows, in situ and in vivo, the threadlike structure (thick arrows) and corpuscle (arrow) inside the lymphatic vessel.

1-2: Stereomicroscope observation

The stained lymphatic tissues, in vivo and in situ, were observed with stereomicroscope (SZX 12, Olympus) and were recorded with a CCD camera (DP70, Olympus). Fig. IA is a typical stereomicroscope view of a lymphatic vessel near the caudal vena cava of a rat. For clear observation, a piece of black paper was put under the lymphatic vessel in order to isolate the vessel from its complicated surroundings. Halogen lamp was used, and optical fibers with diameters of 3 mm to illuminate the target area. The illuminating direction was about 10° from the horizontal plane (polar angle = 80°), and the azimuthal angle was about 45° from the lymphatic vessel line. Fine tuning the direction of illumination was important for detecting the barely visible threadlike structures, so the direction was adjusted by pressing the optical fiber, which was held by a micro-manipulator, slightly. Once the target structure had been detected, the angle was adjusted by fine tuning to achieve the best contrast. A more magnified view of a section of the lymphatic vessel in

Fig. IB that includes a valve and a corpuscle is shown in Fig. 1C.

Fig. IA shows a typical stereomicroscope view of a lymphatic vessel near the caudal vena cava of a rat. Fig. 1 is a novel threadlike structure (NTS) inside a lymphatic vessel of a rat in situ. (A) A typical image of a lymphatic vessel (outlined with a dashed line) under a stereomicroscope. A valve (double line arrows) and capillaries (arrow heads) in fats under the vessel wall are visible. The NTS is not observable. Bar, 100 μm. (B) The

NTS (thick arrows) in situ is disclosed in a lymphatic vessel by using the fluorescence of nanoparticles. Three corpuscles (arrows) connected by the NTS are shown together with a valve. Bar, 50 μm. A magnified view is presented in (C). The corpuscle looks reddish due to the high concentration of nanoparticles.

1-3: Inverted microscope observation

As described above, the rats were anesthetized with urethane (1.5g/kg) administered intraperitoneally. Under deep anesthesia the abdominal sides of the rats were incised, the lumbar nodes near the caudal vena cava were located and exposed by removing the surrounding peritonea and fats. A piece of a lymphatic vessel similar to the specimen depicted in Fig. IB was taken, fixed with neutral buffered formalin (NBF 10%), put on a slide, and examined under an inverted microscope (Olympus 1X71).

For the specimen, a differential interference contrast image was shown in Fig. 2A and a nanoparticle-fluorescence image was shown in Fig. 2B. The differential interference contrast image in Fig. 2A shown the threadlike structure made partially protrude from the lymphatic vessel. One end of that vessel had been torn off using a sharp needle, but the debris had not been removed. A fluorescence image of the same specimen is shown in Fig.

2B. Even after having been fixed with NBF, the threadlike structure is very bright clearly proving its existence, due to the fluorescence of the uptaken nanoparticles. Fig. 2B vividly shows the NTS (thick arrow) inside the lymphatic vessel (dotted arrow). The upper side of the vessel wall was opened a little bit to let the NTS protrude. Bars, 50 μm. The specimen depicted in Fig.2A was stained with 0.1% (w/v) acridine orange, and then were observed with the inverted microscope. The result was shown Fig.2C where the nuclei (arrow heads) in the protruding part were revealed by using acridine orange staining. Bar, 20 μm.

1-4: CLSM observation of cryo-sectioned specimen

A piece of a lymphatic vessel prepared in Example 1-1 was taken, fixed with neutral buffered formalin (NBF 10%), and observed with a Zeiss LSM 510 CLSM instrument in fluorescence mode. The confocal laser scanning microscope image of a cross section of the novel threadlike structure (NTS) was shown in Fig. 3A. The NTS is brighter than the vessel wall because it absorbs more nanoparticles than the wall does. In a less sensitive image (Fig. 3B), the lymphatic vessel is no longer visible, and there appear dark holes in the NTS. The dark holes are the locations of nuclei. The nanoparticles were taken up by the cytoplasm but not by the nuclei (11).

A piece of a lymphatic vessel was taken, and fixed with neutral buffered formalin (NBF 10%), stained with l%(w/v) acridine orange, and observed with CLSM. The merged image of acridine orange and the nanoparticle showed nuclei by green color at the dark holes and in the vessel wall (Fig. 3C). The specimen was prepared in a cryo-section with a thickness of about 6 μm.

Fig. 3 is confocal laser scanning microscope images of a cross section of a novel threadlike structure (NTS). (A) The NTS (thick arrow) is brighter than the vessel wall (dotted arrow). (B) In a less sensitive image the NTS (thick arrow) shows dark holes (arrow heads), but the surrounding vessel wall is not visible. Bar, 10 μm. (C) Staining by acridine orange reveals the positions of the nuclei (arrow heads) in the NTS and the position of the lymphatic vessel wall (dotted arrow). The nanoparticles were not absorbed by the nuclei. Bar, 20 μm.

1-5: CLSM observation of cryo-fixed specimen A piece of a lymphatic vessel prepared in Example 1-1 was taken, and fixed with neutral buffered formalin (NBF 10%). The fixed sample was packed into a rivet that was mounted onto a slotted metal stub, and plunged into liquid nitrogen for cryo-fixation for 20 seconds. Frozen specimens were placed in a cryo-chamber of a cryo-transfer system (CTl 500, Oxford Instruments, Oxon, UK) maintained at -170 ^° C. The cryo-sectioned specimen was observed with a Zeiss LSM 510 CLSM instrument. Confocal laser scanning microscope images of cryo-sections of a corpuscle inside a lymphatic vessel with a valve are shown in Fig 4.

The differential interference contrast (DIC) image was shown in Fig. 4A. The fluorescent image was shown in Fig. 4B. The merged image of DIC and the fluorescence of the nanoparticles was shown in Fig 4C. The valve (double line arrow) is in the middle of the lymphatic vessel (dotted arrows). The corpuscle (arrow) is on the right, and the NTS (thick arrow) is on the left. There are four sinuses (arrow heads) in the corpuscle. In the magnified view of the corpuscle in Fig. 4C, two large holes with spherical bodies inside are

clearly seen. These are thought to be sinuses through which some liquid and granules flow, and to be deeply involved in the physiological functions of the novel structure.

1-5: Analysis of size of the Bonghan duct and corpuscle The stereomicroscopic images of thirteen rats according to Example 1 were recorded with CCD camera. The thickness and size of the threadlike structure and the corpuscle were measured using the scale bar on the image, and then calculated to obtain the mean. The thickness of a threadlike structure varied widely, with the thick and the thin parts having average thicknesses of 52±30 μm and 19±9 μm, respectively. The corpuscles had various oval shapes; with an average size of 0.52 mm x 0.15 mm, and the average thickness of the lymphatic vessels was 0.24±0.07 mm. The number and the sizes of the corpuscles varied widely from one subject to another.

EXAMPLE 2 Hematoxylin and Eosin Studies on Threadlike and Corpuscular Structures

In addition to the morphological studies presented in the text, hematoxylin-eosin staining was performed to study histological property of the novel threadlike and corpuscular structures. Fig. 6A showed a cross section of a lymphatic vessel with its valve and corpuscle. The lower part (inside the dotted boundary) is a corpuscle. The upper part is thought to be a piece of very thick threadlike structure attached to the corpuscle. A part of the magnified view is shown in Fig. 6B, where various types of cells, including red blood cells, are seen. The nature of these cells has yet to be studied.

Fig. 6C and D show another section of the corpuscular structure from the same

specimen, but the valve is absent. Here, there are three spherical objects which may flow through the lumens. The presence of red blood cells may also be related with the hematopoeitic function of the new tissue, but its precise nature has yet to be studied. Fig. 6E shows a lymphatic vessel with a corpuscle and a thick threadlike structure. This Fig. is given for illustration purpose: if a cross section were made along the dashed line across the vessel, the produced image would be similar appearance to those in Fig. 6C and D.

Finally, the lymphocytes were captured in certain sections of the lymphatic vessel, as shown in Fig. 6F. A threadlike structure is caught in the lower corner. Its nanoparticle- fluorescence image and its magnified image are shown in Fig. 6G and H. it can be again confirmed that nuclei do not take up the nanoparticles.

The reference as indicated above are as follows:

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[in Japanese].

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on the surface of the liver. J. Intl. Soc. Life Info. Sci. 22, 460 (2004).

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(N. Y. Academic Press, N. Y, 1972), pp. 189-202.

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299, 599 (2002).

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