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.

Distribution of Mast Cells and Locations, Depths, and Sizes of the Putative Acupoints CV 8 and KI 16.

The anatomical locations and sizes of acupuncture points (APs) are identified in traditional Chinese medicine by using the cun measurement method. More precise knowledge of those locations and sizes to submillimeter precision, along with their cytological characterizations, would provide significant contributions both to scientific investigations and to precise control of the practice of acupuncture. Over recent decades, researchers have come to realize that APs in the skin of rats and humans have more mast cells (MCs) than neighboring nonacupoints. In this work, the distribution of MCs in the ventral skin of mice was studied so that it could be used to infer the locations, depths from the epidermis, and sizes of three putative APs. The umbilicus was taken as the reference point, and a transversal cross section through it was studied. The harvested skins from 8-week-old mice were stained with toluidine blue, and the MCs were recognized by their red-purple stains and their metachromatic granules. The three putative APs, CV 8 and the left and the right KI 16 APs, were identified based on their high densities of MCs. These findings also imply that acupuncture may stimulate, through MCs, an immune response to allergic inflammation.

Temporal Change of Alcian Blue-Stained Primo Vascular System in Lymph Vessels of Rats.

This study aims to investigate the temporal change of a vascular system now known as the primo vascular system (PVS). We used Alcian blue (AB) dye for imaging the distribution of the PVS in lymphatic vessels. The target lymph vessels were chosen as they are easily accessible from the skin, and long-term observation is possible with intact physiological conditions due to a minimal surgical procedure. AB solution was injected into the inguinal lymph node and the target lymph vessels were located along the superficial epigastric vessels. The imaging system allowed processing for extraction of images showing changes in the AB intensity of the visualized PVS components. This newly developed procedure can be used for further study on various dynamic processes of PVS in lymph vessels.

Monitoring the primo vascular system in lymphatic vessels by using window chambers.

This study aims to develop a window chamber system in the skin of rats and to monitor the primo vascular system (PVS) inside the lymphatic vessels along the superficial epigastric vessels. The PVS in lymphatic vessels has been observed through many experiments under in vivo conditions, but monitoring the in vivo PVS in situ inside lymphatic vessels for a long time is difficult. To overcome the obstacles, we adapted the window chamber system for monitoring the PVS and Alcian blue (AB) staining dye solution for the contrast agent. The lymphatic vessels in the skin on the lateral side of the body, connecting the inguinal lymph nodes to the axillary lymph nodes, were the targets for setting the window system. After AB had been injected into the inguinal lymph nodes with a glass capillary, the morphological changes of the stained PVS were monitored through the window system for up to twenty hours, and the changes in the AB intensity in the PVS were quantified by using image processing. The results and histological images are presented in this study.

Ultrastructure of a Mobile Threadlike Tissue Floating in a Lymph Vessel.

Observations of the primo vascular system (PVS) floating in lymph ducts were reported by various groups. There have been, however, no studies on the ultrastructure of the entire cross section of a primo vessel (PV) inside a lymph vessel with a transmission electron microscope (TEM). In the current study we took the TEM images of a cross section of the PV inside a lymph vessel. We used the Alcian blue staining method for the finding of the target PV in a lymphatic vessel by injecting the dye into the inguinal lymph nodes. The stained PV was harvested together with the lymph vessel and some parts of the specimens were used for studying with optical microscopes. Some other parts were treated according to a standard protocol for TEM. As the results the TEM study revealed the loosely distributed collagen fibers with plenty of empty spaces and the lumens with the endothelial nuclei. It turned out to be very similar to the ultrastructure of the PVs observed on the surfaces of internal organs. It also showed how compactly the PV is surrounded with lymphocytes. In conclusion, the detailed morphological features like the distribution of fibers in the PV were revealed and shown to be similar to another kind of the PV on the surfaces of internal organs.

Observation of a Flowing Duct in the Abdominal Wall by Using Nanoparticles.

The primo vascular system (PVS) is being established as a circulatory system that corresponds to acupuncture meridians. There have been two critical questions in making the PVS accepted as a novel liquid flowing system. The first one was directly to show the flow of liquid in PVS and the second one was to explain why it was not observed in the conventional histological study of animal tissues. Flow in the PVS in the abdominal cavity was previously verified by injecting Alcian blue into a primo node. However, the tracing of the dye to other subsystems of the PVS has not been done. In the current work we injected fluorescent nanoparticles (FNPs) into a primo node and traced them along a primo vessel which was inside a fat tissue in the abdominal wall. Linea alba is a white middle line in the abdominal skin of a mammal and a band of fat tissue is located in parallel to the linea alba in the parietal side of the abdominal wall of a rat. In this fat band a primo vessel runs parallel to the prominent blood vessels in the fat band and is located just inside the parietal peritoneum. About the second question on the reason why the PVS was not in conventional histological study the current work provided the answer. Histological analysis with hematoxyline and eosine, Masson's trichrome, and Toluidine blue could not discriminate the primo vessel even when we knew the location of the PVS by the trace of the FNPs. This clearly explains why the PVS is hard to observe in conventional histology: it is not a matter of resolution but the contrast. The PVS has very similar structure to the connective tissues that surround the PVS. In the current work we propose a method to find the PVS: Observation of mast cell distribution with toluidine blue staining and the PN has a high density of mast cells, while the lymph node has low density.

Comparison of Alcian Blue, Trypan Blue, and Toluidine Blue for Visualization of the Primo Vascular System Floating in Lymph Ducts.

The primo vascular system (PVS), floating in lymph ducts, was too transparent to be observed by using a stereomicroscope. It was only detectable with the aid of staining dyes, for instance, Alcian blue, which was injected into the lymph nodes. Some dyes were absorbed preferentially by the PVS than the lymph wall. It remains a standing problem to know what dyes are absorbed better by the PVS than the lymph walls. Such information would be useful to unravel the biochemical properties of the PVS that are badly in need for obtaining large amount of PVS specimens. In the current work we tried two other familiar dyes which were used in PVS research before. We found that Trypan blue and toluidine blue did not visualize the PVS. Trypan blue was cleared by the natural washing. Toluidine blue did not stain the PVS, but it did leave stained spots in the lymph wall and its surrounding tissues, and it leaked out of the lymph wall to stain surrounding connective tissues. These completely different behaviors of the three dyes were found for the first time in the current work and provide valuable information to elucidate the mechanism through which some special dyes stained the PVS preferentially compared to the lymphatic wall.

Primo Vascular System: An Endothelial-to-Mesenchymal Potential Transitional Tissue Involved in Gastric Cancer Metastasis.

Gastric cancer is the fourth commonest cancer in the world and the second leading cause of cancer-related death. Investigation of gastric cancer metastasis is one of the hottest and major focuses in cancer research. Growing evidence manifested that primo vascular system (PVS) is a new kind of circulatory system beyond vascular and lymphatic system. Previous researches revealed that PVS is a specific tissue between endothelium and mesenchyme and is involved in cancer, especially in tumor metastasis and regeneration. In current study, we investigated the role of primo vessels in gastric cancer metastasis and its possible relationship to vascular vessels formation. Our results indicated that primo vessels were involved in gastric cancer metastasis. We observed blood vessel-mediated metastasis, primo vessel-mediated metastasis, and an intermediate state between them. We deduced that primo vessels may be precursors of blood vessels. These results possibly provided a thoroughly new theoretic development in cancer metastasis.

Hypothesis on the Treatment of Gliomas with Acupuncture at the Primo Node Corresponding to Zusanli (ST 36).

Background: The primo vascular system (PVS) is an anatomical structure that is a network of ducts with fluid flowing in them, which are called primo vessels and correspond to acupuncture meridians, and primo nodes that correspond to acupoints. The PVS' main function is considered to be the maintenance of regenerative homeostasis in human and animal bodies. This system is distributed throughout the bodies of normal animals and develops around and in cancer tissues. This cancer-associated PVS may be a critical metastatic path besides the blood and the lymph vessels. The author of this article proposes a hypothesis on cancer treatment: Injecting anticancer drugs into acupoints according to the pharmacopuncture method can be effective as a result of the flow channels of the PVS. The author considers the acupoint Zusanli (ST 36) and the route of the primo vessels starting from it. This specific PVS route runs along the perineurium of the sciatic nerve, the pia mater, and the arachnoid mater of the spinal cord to the brain. Thus, by injecting a suitable anticancer drug into ST 36, one can deliver the drug into the brain to treat gliomas and other brain tumors. This new drug-delivery method is just one of the new clinical applications that are possible by combining acupuncture and using the PVS. Conclusions: Anticancer drugs for glioma can be injected into the primo node at the acupoint ST 36 to reach the cancer tissue through the PVS in the sciatic nerve, spine, and brain that can avoid the blood-brain barrier.

Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy.

Number Density of Mast Cells in the Primo Nodes of Rats.

Mast cells (MCs) play a major role in allergic reactions. Surprisingly, the acupuncture points have a higher density of MCs compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. We hypothesized that the primo vascular system (PVS), which was proposed as the anatomical structure of the acupuncture points and meridians, should have a high density of MCs. In order to test that hypothesis, we investigated the primo nodes isolated from the surfaces of internal organs, such as the liver, the small and the large intestines, and the bladder. The harvested primo nodes were stained with toluidine blue, and the MCs were easily recognized by their red-purple stains and their characteristic granules. The results showed a high density of MCs in the primo nodes and confirmed the hypothesis. The MCs were uniformly distributed in the nodes. The relative concentration of the MCs with respect to other cells was ∼15%. We divided the sizes of the primo nodes into three classes: large, medium, and small. The number density and the relative concentration of MCs did not show a size-dependence. The current work suggests that the PVS may participate in the immune response to allergic inflammation, which closely involves MCs.

Protocol for Detecting the Primo Vascular System in the Lymph Ducts of Mice.

The primo vascular system (PVS), which is the proposed conduit for the acupuncture Qi, is a complex network distributed throughout an animal's body. However, even with a microscope, it is not easily detectable because of its transparency. Thus, its existence is largely unknown in current anatomy. A convincing demonstration of its existence is needed. The lymph-primo vessel (PV), which is a subsystem of the PVS, is a very effective visual demonstration of the PVS. The lymph-PVS is a mobile threadlike structure floating in lymph ducts that has been observed in rabbits, rats, and mice by several independent teams. The involved techniques are novel and rather complicated; therefore, we have already provided detailed protocols for the surgery; for the injection of the staining dye; and for the detection, extraction, and identification of the PVS in rabbits and rats. However, the mouse is one of the most important laboratory animals used for various biomedical research purposes. For the convenience of researchers who wish to initiate the PVS experiments in mice, we provide a shortened version of the protocol, despite many similarities with previously published protocols. Thus, researcher can easily obtain the samples of the lymph-PVS of mice.

Observation of the Primo Vessel Approaching the Axillary Lymph Node with the Fluorescent Dye, DiI.

The primo vascular system (PVS) floating in lymph fluid has mostly been observed in large caliber ducts around the caudal vena cava and the thoracic duct of rabbits, rats, and mice. But the PVS has not been traced up to the lymph nodes. It has not been established whether the PVS leaves the lymph vessel through the lymph vessel wall or it enters the lymph nodes. Therefore, observing the PVS entering a lymph node, for example, the axillary node, is desirable. In the current work, we traced the PVS approaching up to the surface of axillary node of a rat. The method used for this study was based upon a method that was recently developed to detect the PVS in the lymph duct from the inguinal to the axillary nodes in the skin of a rat by injecting Alcian blue into the inguinal node. However, the Alcian blue blurred near the lymph nodes and tracing the PVS up to the lymph nodes has not been possible. The current method clearly showed the PVS approaching the axillary node.

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.

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.

Electromechanical method coupling non-invasive skin impedance probing and in vivo subcutaneous liquid microinjection: controlling the diffusion pattern of nanoparticles within living soft tissues.

Transdermal drug delivery is the way to transport drug carriers, such as nanoparticles, across the skin barrier to the dermal and/or subcutaneous layer. In order to control the transdermal drug delivery process, based on the heterogeneous and nonlinear structures of the skin tissues, we developed a novel electromechanical method combining in vivo local skin impedance probing, subcutaneous micro-injection of colloidal nanoparticles, and transcutaneous electrical stimulation. Experiments on the nude mice using in vivo fluorescence imaging exhibited significantly different apparent diffusion patterns of the nanoparticles depending on the skin impedance: Anisotropic and isotropic patterns were observed upon injection into low and high impedance points, respectively. This result implies that the physical complexity in living tissues may cause anisotropic diffusion of drug carriers, and can be used as a parameter for controlling drug delivery process. This method also can be combined with microneedle-based drug release systems, micro-fabricated needle-electrodes, and/or advanced in vivo targeting/imaging technologies using nanoparticles.