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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 microm 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.

A threadlike structure in blood vessels that is considered part of the Bohghan duct system was first reported about 40 years ago. This structure has remained elusive since then due to the inability of other researchers to duplicate the original identification. In this study we identified the characteristic features of this threadlike structure in rats by the use of fluorescent microscope imaging of nuclei stained by acridine orange perfusion. The nuclei of the tissue that comprises the structure are rod-shaped, 10 -20 m long, and aligned in a broken-line striped fashion. The novel technique reported here can differentiate the intravascular threadlike structure from the fibrin strings that normally form during perfusion and can confound the observation procedure. The physiological function of the threadlike structure remains to be elucidated; however, further study may provide a new understanding of mammalian biology in general, and of acupuncture and Oriental medicine in particular. Anat Rec (Part B: New Anat) 278B:27-30, 2004.

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A visualizing agent, Trypan blue, was found to be preferentially effective for Bonghan ducts (BHDs) and corpuscles compared to blood vessels or adipose tissues. By using it, we observed a weblike network of BHDs which was in various membrane structures, such as the peritoneum, and omenta. This network of BHDs in the membrane structures was connected to the freely movable BHDs which did not adhere to the surfaces or wrapping membranes of internal organs. In addition, tracing BHDs in adipose tissues became possible because Trypan blue does not stain adipose tissue.

Novel threadlike structures of 20 to 40 microm in diameter were observed running afloat in the cerebrospinal fluid of the brain ventricles and the spinal central canal of a rabbit. We developed an effective in situ staining technique using hematoxylin to visualize the threadlike structure. The presence of the rod-shaped nuclei in the threadlike structure was confirmed by various nucleus specific staining dyes such as 4',6'-diamidino-2-phenylindole, propidium iodide and yoyo-1. The threadlike structure was surrounded by a cellular membrane, whose presence was visualized by using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate staining. The location, diameter, optical transparency and the presence of rod-shaped nuclei in and the surrounding membranes of the threadlike structure were consistent with a nerve Bonghan duct. The Bonghan duct was claimed to be the extension of the physical substance of acupuncture meridians and to be a distinct type of circulatory system present in mammals. Although Bonghan theory has not been reproduced for a long time, recently, some portions of the Bonghan duct network were confirmed in various organs in mammals including blood vessels, lymphatic vessels and enteric organs. The novel threadlike structure in the central nervous system, more specifically in brain ventricles, is one in a series of findings in an attempt to rediscover the Bonghan duct network.

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