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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 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.     

Historical Observations on the Half-Century Freeze in Research between the Bonghan System and the Primo Vascular System

This article provides potential reasons for the past 45-year halt in research between the time of the Bonghan system of Bong Han Kim (B.H. Kim) and that of the primo vascular system (PVS) of Kwang-Sup Soh (K.S. Soh), briefly but more accurately in its history. Over the years, numerous questions related to the Bonghan system and the PVS have arisen, especially from researchers interested in pursuing PVS research: When and how did B.H. Kim's study results on the Bonghan system become known to public? Why did B.H. Kim and his publications disappear after 1966? Why was little study performed on the system for almost 50 years after Kim? Why and how was the research on the system reinitiated in 2002 by Kwang-Sup Soh? Why did the Bonghan system become the PVS? These questions, as well as technical difficulties in identifying the system, have discouraged many researchers from becoming involved in research on the system. The motivation for preparing this article was to remove doubts about the existence of this important organ, which might have been caused by its unusual and unclear historical background, by providing an accurate history.     

肝郁痰凝型乳腺癌的钼靶X线表现及病理分析

目的：研究肝郁痰凝型乳腺癌的钼靶X线影像特点，探讨其病理基础。方法：观察41例中医证型为肝郁痰凝型、术后病理检查结果为乳腺癌患者的钼靶X线影像表现，对其病理学基础进行分析。结果：41例肝郁痰凝型乳腺癌中，乳腺类型以致密型及混合型居多（占78％）。异常血管征及透环征出现频率较高（占80％以上）。腋淋巴结转移出现频率偏低（占12％）。结论：（1）肝郁痰凝型乳腺癌患者，乳腺密度多偏高，可能与较多发生在绝经期前有关。（2）肿瘤周围透亮环征象及异常血管征象出现频率较高，表明此型患者肿块周围脂肪代谢异常较为普遍及血管生长刺激因子分泌较多。（3）腋窝淋巴结转移者所占比例较小，可能是由于此类患者多属于癌变早、中期，相对气血不足或脾肾虚弱等正虚表现不明显。     

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.     

The Primo Vascular System as a New Anatomical System

Traditional Eastern medicine has had a successful existence for a long time and has provided functional paths for curing disease. However, some scientists do not accept acupuncture, primarily because the meridian system lacks a physical anatomical basis. To date, scientific theories have not been able to explain the functional paths used by traditional Eastern medicine to cure disease. According to Western medicine, no known anatomical foundation exists for the meridians and unknown nervous, circulatory, endocrine, and immune mechanisms mediate the effects of acupuncture. In the early 1960s, only one hypothesis was proposed to explain the anatomical basis of the meridians. By using different experimental approaches during the past 10 years, the number of scientific papers that report the discovery of different anatomical and physiological evidence confirming the existence of an anatomical basis for the meridian system has increased. Morphological science is greatly challenged to offer a new biomedical theory that explains the possible existence of new bodily systems such as the primo vascular system (PVS). The PVS is a previously unknown system that integrates the features of the cardiovascular, nervous, immune, and hormonal systems. It also provides a physical substrate for the acupuncture points and meridians. Announcements of the morphological architectonics and the function of the PVS fundamentally changed the basic understanding of biology and medicine because the PVS is involved in the development and the functions of living organisms. We propose a new vision of the anatomical basis for the PVS and the vital energy—called “Qi”—as an electromagnetic wave that is involved very closely with the DNA in the PVS. DNA provides genetic information and it functions as a store of information that can be obtained from the electromagnetic fields of the environment. The PVS is the communication system between living organisms and the environment, and it lies at the lowest level of life. The theory of the PVS could be a good basis for forming a new point of view of Darwin's evolutionary theory. Discoveries in morphological theory—such as discoveries with respect to the PVS—have not been made since the 18th century. For that reason, the PVS needs more attention.     

亚甲蓝在新辅助化疗后乳腺癌前哨淋巴结活检中的应用研究

目的探讨乳腺癌新辅助化疗后单用亚甲蓝行前哨淋巴结活检的可行性及准确性。方法对68例乳腺癌患者先行3～4周期的新辅助化疗,然后单用亚甲蓝行前哨淋巴结活检术,前哨淋巴结活检后均行腋窝淋巴结清扫术(LevelⅠ、Ⅱ),前哨淋巴结及腋窝的淋巴结状况由石蜡连续切片、HE染色进行病理诊断。结果本组新辅助化疗后患者前哨淋巴结检出率为90%(61/68),假阴性率为9%(3/32),准确率为95%(58/61),灵敏度为91%(29/32)。平均每例检出前哨淋巴结2.1枚。患者月经状况、病理类型、肿块的位置、新辅助化疗方案、疗效对前哨淋巴结的检出率及假阴性率均无影响,而化疗后肿瘤的直径及肿瘤脉管内侵犯影响前哨淋巴结的检出率,但不影响假阴性率。患者均未发生变态反应及皮肤坏死。结论单用亚甲蓝可在新辅助化疗后乳腺癌前哨淋巴结活检中准确预测腋窝淋巴结的状况,并且安全可靠,但化疗后肿块的直径及肿瘤的脉管内侵犯可能是影响检出率的因素,需进一步提高检出率并降低假阴性率。     

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.