Putative Primo-Vascular System in Rabbit Placenta

The primo vascular system (PVS) is a very important topic of study nowadays because of their role in transport and regeneration of tissue and in cell migration and cancer metastasis. The PVS was detected in different organs of the rabbit but not in the placenta. In this work, we observe the PVS inside the blood vessels of the placenta for the first time. The main characteristic features of the primo vessels (PVs) from the rabbit placenta were in agreement with the PVS in different organs of animals, including the rod-shaped nuclei and their arrangement.     

Primo Bundles Identified by Microcomputed Tomography in Primo Vascular Tissue on the Surface of Rat Abdominal Organs

BackgroundThe primo vascular system (PVS) is a novel network composed of primo nodes (PNs) and primo vessels (PVs). Currently, its anatomy is not fully understood.ObjectivesThe aim of this study was to elucidate the three-dimensional PN–PV structure.MethodsOrgan-surface PVS tissue was isolated from healthy and anemic rats. The tissues were analyzed by X-ray microcomputed tomography (CT), hematoxylin and eosin staining, and scanning electron microscopy.ResultsFrom CT images, we identified one or more bundles in a PV. In the PN, the bundles were enlarged and existed in isolation and/or in anastomosis. The transverse CT images revealed four areas of distinct intensities: zero, low, intermediate, and high. The first two were considered to be the sinuses and the subvessels of the PVS and were identified in the hematoxylin and eosin–stained PN sections. The enlargement of the PN from anemic rats was associated with an increase in the intermediate-intensity area. The high-intensity area demarcated the bundle and was overlapped with the mesothelial cells. In scanning electron microscopy, the PV bundles branched out, tapering down to a single bundle at some distance from the PN. Each bundle was composed of several subvessels (∼5 μm). Clustered round microcells (1–25 μm), scattered flat oval cells (∼15 μm), and amorphous extracellular matrix were observed on the surface of the PVS tissue.ConclusionsThe results newly showed that the primo bundle is a structural unit of both PVs and PNs. A bundle was demarcated by high CT intensity and mesothelial cells and consisted of multiple subvessels. The PN bundles contained also sinuses.     

Mesothelial Cells Covering the Surface of Primo Vascular System Tissue

The primo vascular system (PVS) is reported to have a periductium composed of cells with spherical or spindle-shaped nuclei and abundant cytoplasm. However, little is known about these periductium cells. In this study, we examined the morphological features of cells covering the PVS tissue isolated from the surface of abdominal organs of rats. By hematoxylin and eosin (H&E) staining, we observed a layer of dark nuclei on the basement membrane at the borders of the sections of primo node (PN), primo vessel (PV), and their subunits. The nuclei appeared thin and linear (10-14 μm), elliptical (8-10 × 3-4 μm), and round (5-7 μm). The borders of the PVS tissue sections were immunostained with a selective antibody for mesothelial cells (MCs). Areas of immunoreactivity overlapped with the flattened cells are shown by hematoxylin and eosin staining. By scanning electron microscopy, we further identified elliptical (11 × 21 μm) and rectangular squamous MCs (length, 10 μm). There were numerous stomata (∼200 nm) and microparticles (20-200 nm) on the surface of the PVS MCs. In conclusion, this study presents the novel finding that the PVS periductium is composed of squamous MCs. These cells tightly line the luminal surface of the PVS tissue, including PNs, PVs, and small branches of the PVs in the abdominal cavity. These results will help us to understand the physiological roles such as hyaluronan secretion and the fine structure of PVS tissue.     

Primo Vessel as a Novel Cancer Cell Migration Path from Testis with Nanoparticle-Labeled and GFP Expressing Cancer Cells

Background/aimRecently, a novel circulatory system, the primo vascular system (PVS), was found to be a potent metastatic route of cancer cells. The aim of the current work is to demonstrate that cancer cells injected into the testis migrate through the primo vessel (PV).Materials and methodsNCI-H460 cells labeled with fluorescent nanoparticles (FNP) or green fluorescent protein (GFP) gene transfection were injected into testicular parenchyma in 24 rats. After 24 hours of injection, the abdominal cavity was investigated via a stereomicroscope, to detect the PVS, and the samples were analyzed histologically with 4′,6-diamidino-2-phenylindole (DAPI) and hematoxylin and eosin.ResultsInjected cancer cells were detected inside the PVS distributed on the abdominal organs. Some were detected inside intestinal parenchyma into which the attached primo vessels (PVs) entered.ConclusionThe results supported the fact that the PVS may be a novel migration path of cancer cells, in addition to the lymphatic and hematogenous routes.     

Isolation and Morphological Features of Primo Vessels in Rabbit Lymph Vessels

Until now, even though intensive research has been dedicated to the primo vascular system (PVS) during these years, no statistical data on primo vessels and primo vessels in lymph flow have been available. Recently, the general morphological features of primo vessels in lymph vessels around the abdominal aorta were identified from microdissections of tissues from New Zealand White rabbits, and with Alcian blue staining, primo vessels in lymphatic vessels could be definitely identified under a digital microscope. The micro-dissected specimens in situ reveal rod-shaped nuclei stained by Acridine orange. The blue-stained nuclei, which were distributed in a broken-lined stripe, formed a tube structure of about 20 μm in diameter. The distance between the nuclei of two cells on neighboring aligned stripes, which is also the diameter of the micro tube, was measured to be about 5～10μm. The average length of the primo vessels was 2.4 mm, with the longest being 5.6 mm. The average size of the primo vessel was 50 μm, and the average diameters of the primo and the lymph vessels were 26.0 μm and 258.5 μm, respectively. Occasionally, without the use of Alcian blue staining, milk-white transparent primo vessels were observed floating in lymph vessels. Thus, we suggest that the PVS might also have an important function connected with the lymph system. We also expect the traditional Korean meridian system to leave its invisible world during the last thousands of years and soon enter the visible scientific world.     

Primo Vascular System in the Subarachnoid Space of the Spinal Cord of a Pig

The primo vascular system was recently observed in the central nervous systems of rabbits and rats, but no investigations in large animals have been reported. In the present work we found a putative primo vascular system in the spinal cord of a pig. We obtained spines from four healthy pigs and fixed them with paraformaldehyde. The primo vessels were expected to lie in the subarachnoid space between the pia mater and the arachnoid mater. The composite of three membranes (the pia, the arachnoid, and the dura maters) wrapping the spinal cord was peeled off, isolated from the spine, and put on a slide glass. This composite was stained with 4′,6′-Diamidino-2-phenylindole (DAPI) and phalloidin to show the nuclei and the f-actin, respectively, in the cells of the primo vessels. We observed eleven pieces of the putative primo vessels in the subarachnoid space of the spines at the thoracic spinal nerve area. They had the typical rod-shaped nuclei distributed in a broken line, and f-actin signals around nuclei. The lengths of the nuclei were 12–15 μm, and the thicknesses of the primo vessels were 8～20 μm, which were consistent with other primo vessels that had been observed in the various organs of rabbits, rats, and mice. In addition, we observed branching of the primo vessels, which is again an expected result from previous works. In conclusion, a primo vessel was observed in the subarachnoid space of the spinal cord of a pig. This was the first observation of a primo vessel in a large animal, and the staining method used to observe the primo vessel in a fixed sample was newly developed in this work.     

Study of the primo vascular system utilizing a melanoma tumor model in a green fluorescence protein expressing mouse

A melanoma tumor is a representative malignant tumor. Melanoma tumor growth involves vigorous angiogenesis around the tumor and a vasculogenic-like network inside an aggressive tumor. Primo vessels (PVs) are also found on the surface of the tumor and coexist alongside blood vessels (BVs), and sometimes within the BVs. We hypothesized that the primo vessels system plays a significant role in regulating the development of a melanoma tumor, and therefore has a tight coupling with BVs and angiogenesis. To prove this hypothesis, we developed a murine melanoma model by inoculating melanoma cell lines into the abdominal region. We used a green fluorescent protein (GFP) expressing mouse as a host to distinguish the endogenous source of the tumor PVs. We found strong formation of PVs on the tumor that coexisted with BVs and expression of GFP. PVs also had a tight coupling with adipose tissues, especially with white adipose tissue. These data suggest that the PVs of an induced melanoma tumor evolve endogenously from the host body and may be highly related to BVs and adipose tissue. This model of PVs in an overexpressing GFP mouse is a useful system for observing PVs, primo nodes, and primo vessel networks, and has potential to be developed as a model for examining novel treatments for cancer metastasis.     

Visualization of the Primo Vascular System Afloat in a Lymph Duct

Because of the potential roles of the primo vascular system (PVS) in cancer metastasis, immune function, and regeneration, understanding the molecular biology of the PVS is desirable. The current state of PVS research is comparable to that of lymph research prior to the advent of Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). There is very little knowledge of the molecular biology of the PVS due to difficulties in identifying and isolating primo endothelial cells. Present investigations rely on the morphology and the use of differential staining procedures to identify the PVS within tissues, making detailed molecular studies all but impossible. To overcome such difficulties, one may emulate the explosive development of lymph molecular biology. For this purpose, there is a need for a reliable method to obtain PVS specimens to initiate the molecular investigation. One of the most reliable methods is to detect the primo vessels and primo nodes afloat in the lymph flow. The protocols for observation of the PVS in the large lymph ducts in the abdominal cavity and the thoracic cavity were reported earlier. These methods require a laparectomy and skillful techniques. In this work, we present a protocol to identify and harvest PVS specimens from the lymph ducts connecting the inguinal and the axillary nodes, which are located entirely in the skin. Thus, the PVS specimen is more easily obtainable. This method is a stepping-stone toward development of a system to monitor migration of cancer cells in metastasis from a breast tumor to the axillary nodes, where cancer cells use the PVS as a survival rope in hostile lymph flow.     

A Primo Vascular System Underneath the Superior Sagittal Sinus in the Brain of a Rabbit

The primo vascular systems (PVS) observed in the central nervous system have been limited to the ones floating in the cerebrospinal fluid. In those experiments, it was difficult to obtain the same results because the PVS was not fixed in a given anatomical position. In the current work, we report a finding of a PVS in a well-defined location, namely, underneath the superior sagittal sinus in the sagittal fissure, so that repetition of the experiments is possible. This provides a cornerstone for PVS research because the lack of reproducible sample-taking hindered a deeper study of the PVS, such as RNA sequencing or RNA microarray. This obstacle can be overcome through the discovery in the current work. This PVS showed characteristics of the PVS observed in other organs. It showed the bundle structure of subvessels, the parallel distributions of F-actins, and the rod-shaped nuclei. Furthermore, it had a primo node in front of the confluence of sinuses above the pineal body. It had branches shooting off from the main primo vessel in the subarachnoid space toward the cerebral hemispheres. The results indicate that this PVS underneath superior sagittal sinus has proper features to function as a flowing channel.     

Production and Characterization of Monoclonal Antibodies Against Primo Vascular System of Rat

BackgroundThe primo vascular system (PVS) has been difficult to detect due to its small diameter and translucent features of the threadlike network. Thus, contrast-enhancing dyes including Alcian blue, Trypan blue and Janus green B had to be used for finding and taking out PVS from rat and mouse.ObjectiveGeneration of monoclonal antibodies (mAbs) against PVS of rat was intended to use as a detector for PVS and a biological tool for functional study of PVS.Materials and methodsPrimo vessel (PV) and Primo node (PN) were isolated from organ surfaces of rat and then their proteins were isolated and injected into mouse as an immunogen. The classical traditional method was applied for production of mAbs against PVS. The various techniques, such as cell fusion, screening of hybridoma, ELISA, Western blotting (WB), immunofluorescence microscopy (IF), and limiting dilution, were used to generate mAbs against PVS.ResultsAmong 16 mAbs generated, 4 representative mAbs were characterized with their specificities in ELISA, WB, and IF. α-rPVS-m1-1 and α-rPVS-m4-6 had strong binding affinities to PVS in both ELISA and WB but did not show specificities in IF at all. On the contrary, α-rPVS-m3-2 and α-rPVS-m3-4 almost did not respond in WB but had strong binding affinities in ELISA and specificities in IF. Two mAbs stained predominantly at extra cellular matrix and cell membrane of PVS of rat in IF, and they were able to discriminate PVS from blood vessel (BV) and lymphatic vessel (LV).Conclusions4 representative mAbs against PVS of rat were characterized by ELISA, WB, and IF. α-rPVS-m3-2 and α-rPVS-m3-4, which had strong specificities in IF, can be used as a tool in discriminating PVS from other similar tissues and in elucidate biological function of PVS.     

Protocol for the Observation of the Primo Vascular System in the Lymph Vessels of Rabbits

Molecular-level understanding of the structure and the functions of the lymphatic system has greatly enhanced the importance of this second circulation system, especially in connection with cancer metastasis and inflammation. Recently, a third circulatory system, the primo vascular system (PVS) was found in various parts of an animal's body, especially as threadlike structures floating in the lymphatic flow in lymph vessels. Although the medical significance of this emerging system will require much work in the future, at present, several important suggestions in connection with immune cells, stem cells, and cancer metastasis have already appeared. Experiments to observe the PVS in the lymph vessels near the caudal vena cava of rabbits and rats have been performed by several independent teams, but reproduction requires considerable skill and technical know-how. In this article, we provide a detailed protocol to detect the PVS inside the lymph vessels of a rabbit. Detection and isolation are the first steps in unraveling the physiological functions of the PVS, which awaits intensive research.     

Primo Vascular System Floating in Lymph Ducts of Rats

An epoch-making development in the gross anatomy of the lymph system has emerged: the observation of the primo vascular system (PVS), which is a threadlike structure floating in lymph ducts. The PVS, which was proposed as the conduit for the acupuncture Qi, is a complex network distributed throughout an animal's body. The lymph-PVS, which is a subsystem of the PVS, is one of the most convincing visual demonstrations of the PVS. Because its existence is not easily demonstrated, even with a microscope, due to its transparency, in current anatomy its existence is largely unknown despite its potential significance in physiology and medicine. The lymph-PVS has been observed in rabbits, rats, and mice by several independent teams. Because the involved techniques are rather complicated, we provide detailed protocols for surgery, for injection of the staining dye, and for detection, extraction, and identification of the PVS in a rat.     

Effective Isolation of Primo Vessels in Lymph Using Sound- and Ultrasonic-wave Stimulation

The effects of stimulation with sound and ultrasonic waves of a specific bandwidth on the microdissection of primo vessels in lymphatic vessels of rabbit were investigated. The primo vessels stained with alcian-blue dye injected in the lymph nodes were definitely visualized and more easily isolated by sound-wave vibration and ultrasonic stimulation applied to rabbits at various frequencies and intensities. With sound wave at 7 Hz and ultrasonic waves at 2 MHz, the probability of detecting the primo vessels was improved to 90%; however, without wave stimulation the probability of discovering primo vessels was about 50% only. Sound and ultrasonic waves at specific frequency bands should be effective for microdissection of the primo vessels in the abdominal lymph of rabbit. We suggest that oscillation of the primo vessels by sound and ultrasonic waves may be useful to visualize specific primo structure, and wave vibration can be a very supportive process for observation and isolation of the primo vessels of rabbits.     

Primo Vessel Inside a Lymph Vessel Emerging From a Cancer Tissue

Primo vessels were observed inside the lymph vessels near the caudal vena cava of a rabbit and a rat and in the thoracic lymph duct of a mouse. In the current work we found a primo vessel inside the lymph vessel that came out from the tumor tissue of a mouse. A cancer model of a nude mouse was made with human lung cancer cell line NCI-H460. We injected fluorescent nanoparticles into the xenografted tumor tissue and studied their flow in blood, lymph, and primo vessels. Fluorescent nanoparticles flowed through the blood vessels quickly in few minutes, and but slowly in the lymph vessels. The bright fluorescent signals of nanoparticles disappeared within one hour in the blood vessels but remained much longer up to several hours in the case of lymph vessels. We found an exceptional case of lymph vessels that remained bright with fluorescence up to 24 hours. After detailed examination we found that the bright fluorescence was due to a putative primo vessel inside the lymph vessel. This rare observation is consistent with Bong-Han Kim’s claim on the presence of a primo vascular system in lymph vessels. It provides a significant suggestion on the cancer metastasis through primo vessels and lymph vessels.     

Primo vascular system of murine melanoma and heterogeneity of tissue oxygenation of the melanoma

Murine melanoma requires the complex development of lymphatic, vascular, and non-vascular structures. A possible relationship between the primo vascular system (PVS) and the melanoma metastasis has been proposed. In particular, the PVS may be involved in oxygen transport. Vasculogenic-like networks, similar to the PVS, have been found within melanoma tumors, but their functional relationship with the PVS and meridian structures are unclear. Herein, we report on the use of an electrochemical O(2) sensor to study oxygenation levels of melanoma tumors in mice. We consistently found higher tissue oxygenation in specific sites of tumors (n=5). These sites were strongly associated with vascular structures or the PVS. Furthermore, the PVS on the tumor surface was associated with adipose tissue. Our findings suggest that the PVS is involved in the regulation of metastasis.     

Primo Vascular System as a New Morphofunctional Integrated System

The purpose of this review is to describe the methodology, instruments, and subject animals used until now for studies of the meridian (Kyungrak) system and the primo vascular system (PVS). The PVS is observed as an anatomical system distributed in cavities, organs, and tissues throughout the body. We analyzed the most important points of the PVS based on the results obtained until the present. Our main effort has been directed to describing the main thesis relating to the morphological structures and their topography, the functional mechanisms of the PVS, and possible roles of the PVS in pathological processes. The substance of the PVS in all its aspects is as a system covering the whole body and regulating and coordinating the biological processes that are the basis for life. In conclusion, we suggest that the finding of the PVS represents the discovery of a new integrated morphological-functional system.     

Formative Research on the Primo Vascular System and Acceptance by the Korean Scientific Community: The Gap Between Creative Basic Science and Practical Convergence Technology

The purpose of this study was to trace the formative process of primo vascular system (PVS) research over the past decade and to describe the characteristics of the Korean scientific community. By publishing approximately 30 papers in journals ranking in the Science Citation Index (Expanded), the PVS research team actively convinced domestic and international scientists of the anatomical existence of the PVS and its possible application to Korean and Western medicine. In addition, by sharing the PVS observation technique, the team promoted the dissemination and further pursuit of the research. In 2012, however, PVS researchers performed smaller scale research without advancing to a higher level as compared to the early days. The main reasons were found to be the Korean Research and Development policy of supporting creative, small-scale basic research and applied research of Western scientific fields that promised potentially greater success on an extensive scale; the indifference concerning, and the disbelief in, the existence of a new circulatory system were shown by the Western medical community. In addition, the Oriental medical community was apathetic about working with the PVS team. Professors Kwang-Sup Soh and Byung-Cheon Lee were the prime movers of PVS research under difficult conditions. Spurred by their belief in the existence and significance of the PVS, they continued with their research despite insufficient experimental data. The Korean scientific community is not ready to promote the Korea-oriented creative field of the PVS team.     

A New Definition of an Acupuncture Meridian

This article provides a new definition of an acupuncture meridian. It suggests that a meridian consists of a distal tract of tissue that is affected by organ function. In the 1960s, Kim discovered the primo vascular system and regarded the superficial primo vessels as equating to the meridians. Instead, this article suggests that the superficial primo vessels merely underlie the meridians, in that they enable their creation, which is why some meridians are said to occur along the paths of superficial primo vessels. But the meridians themselves do not have a dedicated anatomical structure; instead they are merely tracts of tissue whose normal function is impeded when the related abdominal organ is stressed. It is hypothesized that the organ information is communicated in electrical waves that may travel through the connective tissue sheaths of the superficial primo vessels. Hence, the primo vessels serve as an inadvertent transport for this information, but the organ information is independent of the physiological purpose of the primo vascular system, as are the resultant meridians.     

Critical Review and Comments on B.H. Kim's Work on the Primo Vascular System

Two periods of primo vascular system (PVS) discovery exist. The first one includes the five reports of B. H. Kim made from 1962 to 1965. The second one is from 2002 until the present time and includes reports made mainly by the Seoul National University group using modern methods. The purpose of this article is to describe the claims in B. H. Kim's reports, to comment on the most important points of his claims, and to offer hypotheses for the morphological architecture and the function of the PVS. The PVS integrated the cardiovascular, nervous, and hormonal systems. Thus, the particularities of the various body systems are combined in the PVS. The PVS is not a simple circulatory system like the cardiovascular system. Its influence on all body systems is a combination of not only substances and signals but also energy and information. The primordial PVS is like a matrix for the vascular and the nervous systems, which are formed around the PVS. The PVS is duplicated by the vascular and the nervous systems in the very early stage of body development. This is the reason why the PVS combines the features of the vascular, the nervous, and the hormonal systems. Subsequently, all embryonic body systems have developed, the primordial PVS remains connected to them, but dominates and controls them as the primeval functional system.