Differential Gene Expression by RNA-Seq Analysis of the Primo Vessel in the Rabbit Lymph

For the connectome of primo vascular system, some long-type primo vessels dyed with Alcian blue injected into inguinal nodes, abdominal node, and axially nodes were visualized, which passed over around the vena cava of the rabbit. The Alcian blue dye revealed primo vessels and colored blue in the rabbit lymph vessels. The length of long-type primo vessels was 18 cm on average, of which diameters were about 20–30 μm, and the lymph vessels had diameters of 100–150 μm. Three different tissues of pure primo vessel, mixed primo + lymph vessel, and only lymph vessel were made to undergo RNA-Seq analysis by next-generation sequencing. We also analyzed differentially expressed genes (DEGs) from the RNA-Seq data, in which 30 genes of the primo vessels, primo + lymph vessels, and lymph vessels were selected for primo marker candidates. From the plot of DEG analysis, 10 genes had remarkably different expression pattern on the Group 1 (primo vessel) vs Group 3 (lymph vessel). With Fragments Per Kilobase of exon per Million the cutoff p-value for each gene was < 0.05. Fragments Per Kilobase of exon per Million of the 10 genes such as IGHM, HLA-DRA, HIST1H41, LPL, CD36, SRGN, DGAT2, SNCG, CD48, and GPD1 for primo vessels compared with those of lymph vessels increased twice or thrice. These results suggest that the selected genes could be used for the specific marker to construct primo connectome of circuit system in the rabbit.     

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 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.     

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.     

Primo Vascular System in Human Umbilical Cord and Placenta

The primo vascular system (PVS) has been observed in various animals such as mice, rats, rabbits, dogs, swine, and cow, but not in humans. In this work, we report on the observation of a human PVS on both the epithelial fascia and inside the blood vessels of the umbilical cord (UC). The main morphological characteristics of the primo vessels (PVs) and primo nodes (PNs) from the human UC were in agreement with those of the PVS in various animal organs, including the thicknesses and the transparency of the PVs, the sizes of the PNs, the broken-line arrangement of the rod-shaped nuclei, the sparse distribution of nuclei, and the presence of hollow lumens in the central inner parts of the PNs. It was rather surprising that the human PV was not thicker than the PVs from small animals. The difference between the PVS and blood/lymph vessels was confirmed using immunofluorescence staining of von Willebrand factor, CD31, LYVE-1, and D2-40. The positive expression of the PVS to proliferating cell nuclear antigen, a cell-proliferation marker, was consistent with the recent finding of very small embryonic-like stem cells in the PVS of mice.     

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.     

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.     

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.     

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.     

Motion Properties of the Sanals of the Primo Vascular System under a Magnetic Field

The motion properties of the sanals of the primo vascular system were investigated under a low static magnetic field of 100 Oe. Sanals of about 1 μm were selected and separated from the primo vessels and nodes on a rabbit’s organ surface. The average velocity of five sanals in a physiologic saline solution parallel and perpendicular to the direction of the applied magnetic field was approximately 1.0 pixel/second in random directions, which implies that the rotating motion of sanals with nuclei composed of DNA containing many inorganic magnetic elements such as manganese and cobalt is monotonically weakened by increasing an applied magnetic field.     

Visualization of the Network of Primo Vessels and Primo Nodes Above the Pia Mater of the Brain and Spine of Rats by Using Alcian Blue

By spraying and injecting Alcian blue into the lateral ventricle, we were able to visualize the network of the nerve primo vascular system above the pia mater of the brain and spine of rats. Staining these novel structures above the pia mater with 4′,6-diamidino-2-phenylindole demonstrated that they coexisted in cellular and extracellular DNA forms. The cellular primo node consisted of many cells surrounded by rod-shaped nuclei while the extracellular primo node had a different morphology from that of a general cell in terms of DNA signals, showing granular DNA in a threadlike network of extracellular DNA. Also, differently from F-actin in general cells, the F-actin in the primo vessel was short and rod-shaped. Light and transmission electron microscopic images of the PN showed that the nerve primo vascular system above the pia mater of the brain and spine was a novel dynamic network, suggesting the coexistence of DNA and extracellular DNA. Based on these data, we suggest that a novel dynamic system with a certain function exists above the pia mater of the central nerve system. We also discuss the potential of this novel network system in the brain and spine as related to acupuncture meridians and neural regeneration.     

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.     

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.     

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.     

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.     

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.     

Growth of Microgranules into Cell-like Structures in Fertilized Chicken Eggs: Hypothesis for a Mitosis-free Alternative Pathway

According to Bonghan Kim's theory of anatomical reality for acupuncture meridians, DNA microgranules known as Sanals are key functional components in the primo vascular system (formerly the Bonghan system). To investigate this issue, we developed a new system, an incubator bound to a phase-contrast microscope, in which we cultivated and then observed for 10 hours microgranules taken from 3-day-old chick embryos and from blastoderms of fertilized chicken eggs. With this system, we found that, over time, the microgranules grew in circular patterns to become cell-like structures. In the embryo specimens, we found two distinctive microgranule growths, which developed into cell-like structures over 10 hours. In the first case, a microgranule of about 1.0 μm in size developed into a 3.3-μm-sized cell-like structure, with a pattern of concentric circles. The growth rate of the diameter of the first microgranule was, on average, 0.23 μm/hour. In the second case, a 2.5-μm-sized microgranule developed into a 5.4-μm-sized cell-like structure, which also exhibited a pattern of concentric circles. The average growth rate of the diameter of the second microgranule was 0.31 μm/hour. In the blastoderm specimens from the fertilized chicken egg, we also found three distinctive concentric growths. Interestingly, one of the three blastoderm microgranules grew very quickly, from about 2.5 μm in size to about 5.5 μm in size during 5 minutes of incubation. This was followed by steady growth to about 7.0 μm in size during the next 10 hours of incubation. In the final step of our investigation, we confirmed that the cell-like structures that had grown from the microgranules stained by acridine orange had DNA signals. We believe that the data obtained with our experimental method provide a clue that a mitosis-free alternative pathway for cell formation may, indeed, exist. We also suggest that this new function of microgranules (Sanals) might be related with the acupuncture meridian called the primo vascular system.     

Bonghan Ducts as Possible Pathways for Cancer Metastasis

ObjectiveThe present study has been designed to find a possible new route for the metastasis of cancer cells on the fascia surrounding tumor tissue using a novel technique of trypan blue staining.Materials and MethodsTumor tissues were grown in the skin of nude mice after sub-cutaneous inoculation with human lung cancer cells. Trypan blue was recently identified as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as blood or lymph vessels or nerves.ResultsWe demonstrate that the trypan blue staining technique allows the first visualization of BHDs which are connected to tumor tissues.ConclusionSince BHDs are known to make up a circulatory system corresponding to acu puncture meridians or collaterals, we propose that, in addition to the currently known blood or lymph vessels, BHDs on tumor tissue fascia may be a novel pathway for 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.