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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Homing of the Stem Cells from the Acupoint ST-36 to the Site of a Spinal Cord Injury: A Preliminary Study

Homing of stem cells (SCs) to desired targets such as injured tissues remains a lingering problem in cell-based therapeutics. Studies on the biodistribution of intravenously administered SCs have shown the inefficacy of blood vessels as the homing path because most of the injected SCs are captured in the capillary beds of the lungs. We considered an alternative administration method using the acupuncture meridians or the primo vascular system. We injected SCs at the acupoint Zusanli (ST-36) below the knee of a nude mouse with a spinal cord injured at the thoracic T9-10 vertebrae. The SCs migrated from the ST-36, along the sciatic nerve, the lumbar 4-5, and then the spinal cord to the injury point T9-10. The SCs were not randomly scattered but were rather well aligned like marathon race runners, along the primo vascular system route toward the injury point. We observed the SCs at 1, 3, 6, 9, 12, and 15 hours after injection. The fast runners among the injected SCs took about 6 hours to reach the sciatic nerve, about 9 hours to reach the lumbar 4-5, and about 15 hours to reach the injury point T9-10.     

Effect of electroacupuncture on the expression of PGC-1α and UCP-1in the brown adipose tissue of obese rats

Objective

To explore the effect of peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α) and uncoupling protein-1 (UCP-1) in the brown adipose tissue (BAT) of obese rats in the process of acupuncture treatment for obesity.

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.     

针刺井穴、重灸督脉对6例持续植物状态患者催醒作用的观察

目的：观察针刺井穴，重灸督脉对持续植物状态患者的催醒作用的预试验。方法：6例患者均以十二井穴、水沟、大椎、素髎等为主穴，加用重灸督脉经百会穴的方法来治疗，分别观察治疗前后患者PVS评分的得分情况。结果：6例患者中促醒2例，1例显效，2例有效，总有效率为83．3％。结论：针刺井穴，重灸督脉的方法治疗持续植物状态，在疗效上有一定的优势，表现出较好的研究前景。     

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.     

Fluorescent Nanoparticles for Observing Primo Vascular System Along Sciatic Nerve

The primo vascular system was found in the epineurium along the rat sciatic nerve following subcutaneous injection of fluorescent nanoparticles at the Zusanli acupoint (ST-36). Nanoparticles were injected into the primo-vessel near ST-36 and flowed along the sciatic nerve. Fluorescence revealed a structure in the epineurium that was hardly detectable. Images of the isolated sample stained with 4′,6-diamidino-2-phenylindole were captured using confocal microscopy. These images showed the distinctive nuclei distribution and multi-lumen structure of primo-vessels that differentiate them from lymphatic vessels, blood capillaries and nerves. This study demonstrates a new use for nanoparticles in fluorescence reflectance imaging techniques during in vivo imaging of primo-vessels.     

Therapeutic effects of dichloromethane fraction of Securidaca inappendiculata on adjuvant-induced arthritis in rat

Ethnopharmacological relevance

Securidaca inappendiculata (SI) is a traditional antirheumatic medicine used in China. The present study was designed to investigate the therapeutic efficacy of dichloromethane fraction of SI (SID) at three different doses on adjuvant induced arthritis (AA) rats.

Methods

Arthritis severity was evaluated by arthritic score, body weight loss, paw circumference, histological changes and hyperplasia of lymphatic tissues. Serum samples were collected for estimation of superoxide dismutase (SOD), glutathione (GSH), hydroxy radical (OH·), nitric oxide (NO), malondialdehyde (MDA), N-acetyl glucosaminidase (NAG), sialic acid (SA), alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT). The levels of GSH, MDA, NAG and SA in liver were also assessed. The levels of interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) were determined using ELISA method. Another portion of blood was used for total and differential leucocyte counts.

Results

Administration with SID (at high dose with 100 mg/kg) significantly ameliorated the AA severity, suggested by the modulatory effects on body weight loss, paw swelling, hyperplasia of lymphatic tissues and synovial membrane, neutrocytosis and lymphocytosis. It also decreased levels of NO, MDA and OH·, restored SOD and GSH levels in serum. The abnormal increased levels of AST, ALT, ALP, NAG and SA significantly were reverted (compared with AA rats, P<0.01). A similar result was observed in livers. Levels of IL-1, TNF-α, MCP-1 and VEGF were reduced dramatically by SID too.

Conclusion

The results suggest SID possesses substantial anti-arthritic activity. The therapeutic efficacy may be due to immumodepressive effects, cytokines regulation, increasing membrane stability and antioxidantive activity.     

前荷叶碱对血管紧张素Ⅱ诱导人脐静脉内皮细胞凋亡的影响

目的观察前荷叶碱对血管紧张素Ⅱ(A ngⅡ)诱导人脐静脉内皮细胞(HUVEC s)凋亡的影响,探讨其对HUVEC s的保护作用机制。方法体外培养HUVEC s细胞系ECV 304,以10μm o l/L卡托普利或10、1、0.1、0.01μm o l/L前荷叶碱作用于HUVEC s,30 m in后再加入A ngⅡ1μm o l/L,光镜下观察细胞形态,M TT法观察前荷叶碱对HUVECs活性的影响,比色法测定NO、总一氧化氮合酶(tNOS)、诱导型一氧化氮合酶(iNOS)水平,流式细胞仪测定细胞凋亡率。结果与对照组比较,Ang能明显诱导HUVECs的凋亡(P<0.01),10μmol/L卡托普利及0.01~10μmol/L前荷叶碱可明显改善内皮细胞形态,组织活性明显升高(P<0.05),能增加HUVECs释放NO及生成tNOS(P<0.05),但对iNOS影响不大,使Ang诱导的HUVECs凋亡细胞数明显减少(P<0.05)。结论前荷叶碱通过增加NO生成而抑制Ang诱导的HUVECs凋亡,从而发挥可能的内皮保护功能。     

Effect of sulfated polysaccharides from Laminaria japonica on vascular endothelial cells in psychological stress rats

Ethnopharmacological relevance

Laminaria japonica is a popular seafood and medicinal plant in China. Laminaria japonica is used in traditional Chinese medicine to treat and prevent hypertension and edema.

Materials and methods

The vascular protective activity and mechanism of sulfated polysaccharides were studied in adrenalin-induced vascular endothelial damage in rats after psychological stress (PS). Vehicle (sham and PS groups), sulfated polysaccharide from Laminaria japonica (LP; 1 mg/kg and 5 mg/kg) and enoxaparin sodium (1 IU/kg, reference drug) were all administered for 10 days. Behavioral changes were recorded. Plasma levels of adrenalin, cortisol, monoamine oxidase (MAO), semicarbazide-sensitive amine oxidase (SSAO), formaldehyde, H₂O₂, nitric oxide (NO), endothelin-1 (ET-1), 6-keto-prostaglandin F_1a (6-keto-PGF_1a), and thromboxane B₂ (TXB₂) were measured. Endothelium-dependent relaxation of the thoracic aorta was measured and transmission electron microscopy of aortic vessels was performed.

Results

Adrenalin metabolites in plasma were significantly lower (P<0.01) in rats after LP administration compared with those in the PS groups. The normalized ratios of plasma NO/ET-1 and 6-keto-PGF_1a/TXB₂ were maintained and endothelium-dependent relaxation of the aorta was greatly enhanced after LP treatment (P<0.05). Morphological alterations were observed in vascular endothelial cells (VECs) in PS rats, with a higher number of lysosomes and vague mitochondrial cristae compared with those in the sham group. However, these histopathological changes were markedly alleviated after LP treatment.

Conclusions

This study shows a protective effect of LP on VECs in PS rats. LP can regulate plasma levels of NO, ET-1, and 6-keto-PGF_1a, enhance endothelium-dependent relaxation, and alleviate histopathological changes of lysosomes and mitochondria in VECs. The potential mechanism of LP on VECs in PS rats is related to its function of reducing metabolites of adrenalin.