- Email: [email protected]
Technical Challenges in Current Primo Vascular System Research and Potential Solutions
+
MODEL
J Acupunct Meridian Stud 2016;--(-):--e--
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
REVIEW ARTICLE
Technical Challenges in Current Primo Vascular System Research and Potential Solutions Kyung A. Kang 1,*, Claudio Maldonado 2, Vitaly Vodyanoy 3,4 1
Department of Chemical Engineering, University of Louisville, Louisville, KY, USA Department of Physiology and Biophysics, University of Louisville, Louisville, KY, USA 3 Department Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, and School of Kinesiology, Auburn University, Auburn, AL, USA 4 The Research Department, The Edward Via College of Osteopathic Medicine, Auburn, AL, USA 2
Available online - - -
Received: Sep 19, 2015 Revised: Jan 28, 2016 Accepted: Feb 8, 2016 KEYWORDS Bonghan system; primo vascular system; PVS identification; PVS harvest; optical characterization
Abstract Since Bonghan Kim’s discovery of the Bonghan system (BHS) in the 1960s, numerous reports have suggested that the system is fundamental for maintaining mammalian life. The BHS is a circulatory system independent of the blood or the lymphatic system, forms an extensive network throughout the entire mammalian body, has been reported to be the acupuncture meridian, stores distinct types of stem cells, and appears to have some roles in cancer metastasis. Despite Kim’s first report having been published as early as 1962, research progress has been rather slow mainly because the system is very small and translucent, making it optically difficult to distinguish it from the hemoglobin-rich surrounding tissues. Unfortunately, Kim did not describe in detail the methods that he used for identifying and harvesting the system and the components of the system. In 2000, Kwang-Sup Soh reopened the BHS research, and since then, new and important scientific findings on the system have been reported, and many of Kim’s results have been verified. In 2010, the BHS was renamed the primo vascular system. Nevertheless, good tools to properly deal with this system are still lacking. In this article, we address some of the technical difficulties involved in studying the primo vascular system and attempt to discuss potential ways to overcome those difficulties.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. * Corresponding author. Department of Chemical Engineering, University of Louisville, Louisville, KY 40292, USA. E-mail: [email protected] (K.A. Kang). pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2016.02.001 Copyright ª 2016, Medical Association of Pharmacopuncture Institute. Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
2
K.A. Kang et al.
1. Introduction In 1962, Bonghan Kim [1], a scientist in North Korea, reported a discovery of a new vascular system in mammals, which was soon named as the Bonghan system (BHS). The small size (< 3 mm) and optical transparency of the system were probably the main reasons for its delayed discovery. Kim and his research team [2e5] published four additional reports by the end of 1965, and the content of the reports strongly implied the system’s importance in the mammalian physiology. Kim’s claim of the system being the acupuncture meridian particularly surprised the scientific community. Unfortunately, Kim’s research activities suddenly stopped in 1965 [6] and had undergone a dormant period until KwangSup Soh reopened the research on the system in 2000. By 2002, new scientific results on the system started to appear in various journals, and in 2010, the system was renamed as the primo vascular system (PVS) [6,7]. The BHS/PVS consists of the ducts (vessels), corpuscles (nodes), various fluid-containing cells, and biochemicals that flow throughout the system. The following are the terms used during the Kim’s time and those after the Bonghan system became the PVS, placed side by side: Bonghan system Bonghan duct Bonghan ductule Bonghan corpuscle Sanal (or Bonghan sanal) Bonghan liquor
Primo vascular system (PVS) Primo vessel (PV) Subprimo vessel (Sub-PV) Primo node (PN) Primo microcell (P-microcell) Primo fluid (P-fluid)
Kim also named the subunits of sanal as the sanalosome, sanaloplasm, and sanal membrane, which were not renamed by PVS scientists. A new term “primogenesis” was created in 2012, by the University of Louisville team (Louisville, KY, USA), to define the phenomenon of generating or forming the PVS, while describing the new PVS formation on/in the cancer xenografts. Kim [2,3] classified the system into several subtypes by their locations. As his research progressed and more details on the system were uncovered, his classification became also updated. The subtypes are as follows. (1) Internal (or intravascular): floating inside blood and lymphatic vessels (LVs). (2) External (or extravascular): outside the blood vessels and nerves, or independently. (3) Intraexternal: freely on the surface of internal organs in thoracic/ abdominal cavities. (4) Intraorganic: inside organ walls. (5) Neural: in/on the central and peripheral nervous systems. (6) Superficial: in the corium or subcutaneous tissue. This subtype was to be surrounded by dense blood vessels and was close to nerves. Kim reported the corpuscles of this subtype to be acupoints and the ducts to be the acupuncture meridian. Currently, all subtypes except the external subtype are positively identified [7,8]. After observing the path of a radioactive tracer injected into predetermined Bonghan corpuscles, Kim [2,3] concluded that the subtypes closely communicated with each other and that the system did not appear to be controlled by a single central unit, but by multicompartmentalized units.
As Kim’s publications were reports (not journal publications requiring peer review processes), his findings were to be verified before they were taken as scientific facts. Until now, only some of Kim’s findings have been reexamined and verified, and many are still to be investigated. Since 2000, PVS scientists have also reported new, important findings about the system, and some of the notable scientific findings on the BHS/PVS are as follows. (1) Kim claimed that the superficial BHS was the acupuncture meridian [1e5], and, recently, the Ryu team [8] in Korea colocalized the acupoints and superficial Bonghan corpuscles/PNs in the abdomen of animal models, validating Kim’s claim. (2) After 2000, PVS scientists confirmed it to be independent of the blood or lymphatic system by the differences in microanatomical structures and biomarkers [9]. (3) The BHS/PVS is an extensive network throughout the entire mammalian body [3,7]. (4) The Pmicrocells, which Kim named as sanals (live eggs, in Korean), are now confirmed to be unique types of stem cells, and positively confirmed to be performing nonmarrow hematopoiesis and repairing damaged cells/tissues [5,10e12]. (5) Bonghan liquor/P-fluid contains various biochemicals and cells, particularly those associated with immune responses [2,3,13]. (6) The PVS appears to have a role in the dynamics of cancer metastasis [9,14], which is a new finding after 2000. Although only limited facts on the BHS/PVS have been revealed so far, the nature of these listed above clearly demonstrates the importance of the system in the mammalian physiology. Despite the urgency for a thorough investigation on this important system, there are still serious impeding issues in performing PVS research. The most serious one is lacking scientific tools to properly handle its extremely small size (PV, 20e50 mm; PN < 1500 mm) and optical transparency [1e5], which cause difficulties in identifying and harvesting the system. Kim presented his study results logically with reasonable details, but did not describe the methods for identifying and harvesting the PVS in animal models at a level that future scientists can repeat his studies. New techniques for identifying/harvesting the PVS have been reported since 2000, but they are still not satisfactory for most of the scientists, particularly those who are new to the system, to be able to reliably access the PVS sample to perform in-depth investigation. In this article, in an effort to support the scientific advancement in the system, the authors have analyzed important issues to overcome and attempted to suggest potential techniques to resolve these issues. We hope that other PVS scientists also share their ideas with the PVS community. It should be noted that, the term “PVS scientists” in this article is used to represent the scientists who have been involved in PVS research after 2000. For the scientific terms associated with the system, PVS (not BHS) terms are mainly used to avoid unnecessary confusion.
2. Identifying PVS with nanoparticles For properly performing any scientific research, the target sample needs to be identified first. Frequently, utilizing
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions biomarkers specific for the target sample is helpful, but identifying/developing PVS biomarkers has not been realized yet because of the difficulties in obtaining sufficient amount of PVS samples. Identifying the PVS in the entire body, with the help of appropriate contrast agents, using medical imaging modalities could be a way of obtaining sufficient samples. However, the usual spatial resolution obtainable with these tools would be 100 mm at best [15], which is much bigger than the average PV diameter. Although some advanced modalities such as specially designed magnetic resonance imaging [16e18] and electron or X-ray microscopy [19e21] provide exceptionally good resolution, they are not readily available to most scientists and not practical for real-time sample harvesting from animal models. Therefore, the main technique for identifying the PVS has been optical (stereo) microscopy, particularly for studies involving surgeries in live animals, which is the current situation for most PVS studies. Identification of the PVS has been performed by taking advantage of its natural anatomical structure, and the PV is known to have at least three unique characteristics (Fig. 1), according to Kim [2,3] and later PVS scientists [19e22]: (1) multiple sub-PVs in a single PV (Figs. 1A and 1B), and the inter-sub-PV space filled with fibrous, amorphous substances [3]; (2) rod-shaped nuclei of sub-PV endothelial cells (Fig. 1A) [3]; and (3) pores in the PV external wall (Fig. 1B). The second property is helpful for confirming whether the harvested sample is a PV or not. The first and, particularly, the third properties have been effectively utilized for placing appropriate optical contrast agents into the PV. It is still not confirmed whether these pores exist in the outer membrane of all six PVS subtypes, but this property has been used for identifying the PVS inside the LV and the intraexternal PVS [22e24]. Electron microscopy and X-ray microscopy [19e21] have determined the pore size of the outer wall of the rabbit intraexternal PV to be
3 1e5 mm, and the size of the PV or the pore does not seem to vary significantly among the species studied. According to Kim [3], the interstitial space of sub-PVs (ductules) has a fibrous structure and is filled with amorphous substances. Therefore, if particles of an appropriate size (i.e., smaller than the pore in the PV wall, but similar to or slightly larger than the spacing in the fibrous structure) are applied to the PV from outside, they are expected to get into the PV via the pores and be retained in the interstitial space. If these particles possess an appropriate optical property, then they would generate optical contrast for the PV. Fig. 2 schematically describes the mechanism of identifying the PVS inside an LV (intra-LV PVS) using nano-sized contrast agents. The LV is a good site for identifying the PVS because it has a translucent vessel wall and its fluid is clear, and, therefore, the optically contrasted, intra-LV PVS can be seen through the LV wall. In this procedure, the contrast agent at an appropriate size is first injected into the lymph node connected to the target LV until the LV is filled with the particles (Fig. 2A), and in time, the particles would enter the PV via the pores. The natural flow of lymphatic fluid clears particles from the LV (outside the PV), while the particles that have already entered the PV remain in the interstitial space, providing optical contrast to the PV (Fig. 2B). One such method exploits the unstable nature of Alcian blue at a pH of > 5.6 [25]. Alcian blue is dark green in color, its molecular size is 2e3 nm, and it is frequently used in staining acidic mono- or polysaccharides of biological samples in histological studies [26]. This dye itself, however, does not appear to stain the PVS preferentially to the LV. For the purpose of PVS identification, Alcian blue powder is first dissolved in PBS (pH 7.4); the solution is boiled (possibly to maximize its solubility in PBS) and filtered to remove large particles. The Alcian blue solution is then injected into the lymph node of the animal until the
Figure 1 Properties unique for the PV. (A) PV illustrated in Kim’s third report (slightly modified from [3]; 1-sub-PV; 2- sub-PV endothelial cell; 3-rod shaped nucleus of endothelial cell; 4-PV external layer; 5-nucleus of PV endothelial cell. (B) Schematic diagram of a PV cross section: pores in the PV outermost wall; multiple sub-PVs in a PV; and fibrous nature of PV interstitial space. PV Z primo vessel; sub-PV Z subprimo vessel. Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
4
K.A. Kang et al.
A
B
Sub-PV
Lymphatic vessel
Nanocon trast agent
Pore on PV wall
PV Ly ymphatic flluid flow Figure 2 Schematic diagrams illustrating the identification of the PVS inside an LV using nano-sized contrast agent. (A) PVScontaining LV is filled with nanoparticles, by injecting them into the lymph node. The particles get into the PV via the pores of the PV external vessel wall. (B) The LV is cleared by its natural fluid flow, while the PVS retains the particles trapped in it, generating optical contrast. LV Z lymphatic vessel; PV Z primo vessel; PVS Z primo vascular system; sub-PV Z subprimo vessel.
LV is filled with it [25]. During the process, the dye forms small particles due to its instability in the PBS and lymphatic fluid at pH 7.4. The actual size of the formed Alcian blue particles is not known, but they go through the pore in the PV outer membrane and remain in the interstitial space, generating contrast for the PVS. The dye preparation and injection steps should to be carried out within a short period of time (not clearly defined yet) before larger particle formation and/or precipitation starts. A more controlled and user-friendly method is using nano-sized particles of an appropriate size range and optical contrast against hemoglobin. The University of Louisville team has developed a technique utilizing hollow gold nanoparticles (HGNs) with a color range from green to blue [27]. HGNs were selected because gold is biocompatible and the size and color of HGNs can be controlled well. In a preliminary study using the rat model, particles in the size range of 50e125 nm worked well, with a success rate of 95% [28]. This method has advantages over the Alcian blue method, because the HGNs have a relatively homogeneous size distribution, preparation steps are simpler, and the PVS in the LV becomes visible within 20 minutes after the particle injection. The nanoparticle optical contrast agent used for the purpose of intra-LV PVS identification requires certain properties when used in live animals: (1) it must be nontoxic, biocompatible, and chemically inert because it is to be injected into LVs; (2) the particle surface should be relatively hydrophilic and should not easily aggregate; and (3) it needs to provide a strong optical contrast against the tissue background (i.e., hemoglobin color). According to
our experiments, dark green, greenish blue, or dark blue seems to fit well for the purpose. Up to now, five out of the six PVS subtypes (except the external subtype) have been positively confirmed. The superficial PVS may also need to be given special attention because this subtype is said to be directly related to the acupuncture therapy, and a thorough understanding of this subtype may help in optimizing the therapy. Kim [2] illustrated how the radioactive tracer injected to a Bonghan corpuscle (PN) in the skin moved to other corpuscles via the meridian. Besides the study by Kim, there have been several studies on tracing meridian paths using radioactive tracers or dyes, by injecting them into a particular acupoint (not Bonghan corpuscles or PNs) [29e33]. Since dealing with radioactive tracers is not ideal, the method developed by Lim et al [8] for the colocalization of PNs and acupoints in the abdominal hypodermis region of rats using the threesolution Hemacolor system may be a better approach. The subtype that is most difficult to identify seems to be the external PVS subtype, judging from the lack of scientific articles on this subtype. The external PVS was reported to be covered with connective tissue, according to Kim [3], and, therefore, accessibility to the pores on its external wall is still not known.
3. Harvesting PVS via microelectromechanical system As described in the Introduction, most PVS scientists have already recognized the potential health benefits that may be achieved by appropriately manipulating the nature of
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions the PVS, e.g., regenerating tissues using one’s own Pmicrocells or detecting cancer via the abnormally formed PVS, etc. However, much of the fundamental information about the PVS (e.g., micro- and submicroanatomical structures, biological compositions, biomarkers, physiological functions, the entire PVS map in the body, and communication between the PVS and other organs) still remains to be studied before any translational work can be done. Besides, we still need to verify all of Kim’s findings, which were published 50 years ago. To acquire these basic scientific information, PVS samples need to be available in a reliable manner, but acquiring a sufficient amount of PVS samples has been difficult because of its small size as well as its identification-related issues already described. The sites that may potentially provide a relatively large amount of PVS samples are the blood and LVs because the PVS runs inside them and they are long. Out of the two, LVs are better candidates, as mentioned earlier. Identification of the intra-LV PVS can be performed more easily in live animals because the floating motion of the PVS in the lymphatic fluid helps identify it better. Once the LV loses its fluid and becomes flattened, the PVS easily sticks to the LV inner wall. Live animals, however, breathe and the target LV also moves with the breathing motion, which makes the harvesting tasks more challenging. Therefore, keeping the target LV still during the breathing motion may be helpful (e.g., fixing the LV on a solid surface). Next, retrieving the PVS from the LV requires extremely delicate microsurgical techniques with good microsized tools. A few medical tools, such as micro-extra-fine surgical knives and grippers (e.g., Excelta, Buellton, CA, USA; nanoScience Instruments, Phoenix, AZ, USA), are currently available for this type of study, although some of them can be expensive and may still need to be tailored for PVS research. The tip of the atomic force microscope may potentially serve as a microknife. A suggested method for the intra-LV PVS, once it is identified, is as follows: make small incisions at two positions on the LV, depending upon the desired amount of PVS samples; then insert the tips of two sets of microgrippers (e.g., nanoScience Instruments) into the two respective incision sites of the LV, to hold the two ends of the PVS; cut the LV along its length between the two incision sites; and cut the two ends of the PVS (outer sides of the grippers) and retrieve the freed PVS by lifting the grippers. One might suggest to first harvest the PVS-containing LV and then retrieve the PVS from it, in order to simplify the process. However, when the LV is cut without holding the two ends of the PVS, the PVS tends to curl up inside the LV because of its elastic nature. Fig. 3 shows the PVS optically contrasted by HGNs, before (Fig. 3A) and after (Fig. 3B) the LV-containing PVS was cut at its lower part, and the PVS is shown to curl up after the LV was cut. In addition, if PVS retrieval is not performed immediately after the LV is cut, then the PVS may stick to the inner wall of the LV, which makes it difficult to harvest the PVS. Since the entire procedure involved in the PVS harvest requires precise operation due to the small target sample size, if the procedure is performed by a micromanipulator based on the microelectromechanical system (MEMS) technology, human errors may be minimized. It is also
5
Figure 3 A PVS identified inside an LV, using HGNs. (A) The PVS contrasted by the HGNs can be seen inside the LV. (B) After the lower end of the PVS bearing LV is cut, the PVS is curled up in the LV. HGN Z hollow gold nanosphere; LV Z lymphatic vessel; PN Z primo node; PV Z primo vessel; PVS Z primo vascular system.
highly desirable to have microliter-sized P-fluid collectors and/or microinjectors, utilizing MEMS technology, for the P-fluid analysis and applying biochemicals into the PVS.
4. P-microcell retrieval by cell sorting techniques Kim [4] reported that there are unique, self-regenerating cells (P-microcells/sanals) inside the PVS, particularly in the PNs [5], and these cells are now positively confirmed to be stem cells [11,12]. A thorough understanding of Pmicrocells and their roles would, therefore, be very important for the stem cell science and regenerative medicine, and retrieving these cells from PNs in a reliable manner is the first step toward it. This may be done, first, by mechanically or enzymatically disintegrating the outer layer of PNs to obtain all cells inside them, and then separating the P-microcells from other cells. Since the constituents or properties of the PN membrane are still not clearly known, development of an optimized process for removing the membranes may have to wait until they are well characterized. After the membranes are removed, P-microcells need to be separated. Over the past decade, cell-sorting techniques have been advanced significantly because detecting circulating cancer cells has been emphasized as an important research topic for early cancer diagnosis. The Pmicrocell is characterized by its small size, 1e3 mm
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
6 [10e12], while most cells are in the range of 10 mm. Therefore, P-microcells may be separated from the cell population in PNs by flow cytometry, either using the difference in cell size [34] or by fluorescence [35] after labeling the cells with fluorophores via antibodies against stem cell biomarkers that P-microcells express [10,36]. For PVS studies, it may also be advantageous to use micro-sized cell-sorting systems [37,38] instead of using a regular flow cytometer, since PNs are very small, and the cell volume and number in a PN are very small.
5. Optical microscopy for PVS characterization Optical microscopy was one of the most important methods used in the discovery of the BHS in the 1960s, and rediscovery of the PVS in the 2000s [39], although it was used mainly for PVS identification instead of characterization. If it is to be used for PVS characterization, the micro- and/or submicrostructural components and translucent optical property of the PVS, however, present serious challenges. In the visible light transmission and reflection microscopy, even for advanced confocal devices, the lateral image resolution would not exceed 180 nm [40], requiring substantially enhanced resolution [41] for detailed structural characterization of the PVS. Optical resolution up to the submicron level may, however, be achieved via the annular illumination to increase the intensity of diffraction fringes, and via the coherent illumination of small (compared to wavelength) objects to
K.A. Kang et al. allow the image edge to become sharper, thus increasing the resolution [42e46]. Minimizing the light spot size on the sample and reducing the stray light can also improve spatial resolution and contrast: reducing the spot dimension raises the irradiance of the sample and increases the contrast by increasing the interaction with small particles in the sample. The photon density of the system modified by the Auburn University team (Auburn, AL, USA) became approximately 150 times higher than that of the conventional Nikon darkfield condenser (Eclipse E400, Oil 1.43-1.20, Japan). This increased illumination intensity raises the optical difference between tiny light-scattering particles and their background. By doing so, all particles larger than 90 nm, including small DNA granules and P-microcells, can be resolved [42]. A high-output numerical aperture of an illumination system allows utilizing high numerical apertures of the microscope objective and produces highly luminous super-resolution images. Figs. 4 and 5 are images with significantly enhanced resolution and contrast, taken by the Auburn University team with proper adjustments described above. Fig. 4 shows an image of an intraexternal PN connected to a PV [3], which was harvested from the surface of the rat intestine. PNs are usually linked to the PVs at their both ends, and in some cases, several PVs are connected to a single PN. This particular PN was, however, connected to the PV at its one end only (Figs. 4A and 4B). Figs. 4B and 4D are enlarged images of the rectangular sections in Figs. 4A and 4C, respectively. The PVS sample was treated with acridine
Figure 4 Optical images of a nonfixed, intraexternal PVS from a rat, stained with acridine orange. (A) Overall view of the PVS. (B) A magnified image of the part connecting the PN and PV in Fig. 4A. (C) A magnified image of the branched PV, shown in the dotted rectangle in Fig. 4A. (D) A further magnified view of the PV shown in the dotted rectangle in Fig. 4C. Its cell nuclei are shown with arrows. The scale bars in A, B, C, and D are 500 mm, 200 mm, 100 mm, and 50 mm, respectively. PN Z primo node; PV Z primo vessel; PVS Z primo vascular system. Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions
7
Figure 5 A transverse sectional view of a small PN. (A) In the brightfield mode, basophilic granules are labeled with black stars. (B) The same section photographed in the darkfield mode. Vacuoles are indicated by white arrows, intravascular space by a white arrow head, cluster of granules by a black arrow, a granule in the small lumen by a black arrow head, the largest lumen by a white star, and the interrupted portion of lumen connected to a vacuole by a dotted white arrow. C Z capsule; L Z lumens; PN Z primo node.
orange, which is used as a nucleic-acid-selective fluorescent cationic dye. The staining reveals cells fluorescing with green and red light: the light at a wavelength of 502 nm excites the acridine orange/DNA complex that emits at 525 nm (green). When it interacts with RNA, excitation shifts to 460 nm and emission to 650 nm (red) [47]. In these images, the details of this PVS may be very clearly seen: the PN has an oval shape with dimensions of 0.85 1.1 mm2 and the PV length is 1.25 mm. The diameter of the middle part of the vessel is w140 mm. The apical end of the vessel is branched into two smaller vessels. The branched vessels are connected to other nodes (not shown here). The green and red colors may be interpreted as DNA and RNA of cell nuclei, but acridine orange also accumulates and emits red light in mast secretory granules and other cellular acidic compartments [48,49]. In Fig. 4D, the red color is emitted from the cell cytoplasm rather than from the cell nuclei. The nuclei of these cells (shown by arrows) are green. These particular cells represent w5% of the entire cell population. Microscopic observation of the mast cells under the acupuncture meridian lines of humans and rats revealed that the mast cells were more concentrated under the meridian lines in comparison with their control areas. Therefore, the red-stained cells in the figure may be mast cells [50e53]. According to this result, the mast cells represent w5% of acridine orange-stained cells in the PVS, which is close to 4%, as quantified by Lim et al [53], but less than 20%, as reported by Kwon et al [13]. The difference may be due to variations in techniques and experimental conditions. Fig. 5 illustrates the transversal section of a PN in the bright and dark field modes (Figs. 5A and 5B, respectively). The walls of sub-PVs are smooth, single layered, and uninterrupted, except for the wall of the lumen labeled by the white star. There is a small opening in the wall that leads to a vacuole (dotted white arrow). Some sub-PVs run very close to each other, with a small intravascular space between them (white arrow head). The section also shows randomly scattered single cells and granules, and some granules are in clusters (black arrow). These features shown in the optical image of the PN cross section agree with those described by electron and light microscopy in general [22,54].
As illustrated here, optical microscopy with proper adjustment can be utilized effectively for PVS characterization in a micron/submicron level.
6. Studying cancer primogenesis via fluorescent protein Most PVS studies performed up to now have focused on the identification and basic characterization of the system probably because the system is still new. However, there have been interesting preliminary study results on the behaviors of the PVS in/adjacent to cancerous tumors (cancer PVS), at least for six different cancer xenografts. Although this topic is not associated with basic characterizations of the normal PVS, we included it in the article because cancer affects many people, and a thorough understanding of the cancer PVS may lead to better management of the disease. The cancer xenografts studied up to now are: lung (NCI-H460) and ovarian (SK-OV-3) cancer [9,14], cutaneous melanoma (B16 F10) [55], breast cancer (MDA-MB231), gastric cancer (MKN12) [56], human leukemia (U937) [57], and also human breast cancer mimicking cell line 4T1 [58]. The PVS in/on normal tissues or organs is usually neither very densely populated nor easily observed. The cancer PVS, however, has been shown to form at a higher density in/around the tumor. In addition, cancer PVS has been shown to participate in cancer cell transport from the primary to the secondary tumors [9,14]. To learn how the cancer PVS proliferates, Heo et al [55] used a combination of green fluorescent protein (GFP)expressing host animals and nonfluorescing human skin cancer cell line, and the result showed that GFP PVS was formed inside the tumor. The Soh team [56] used a GFPexpressing cancer cell line in nonfluorescing animal models and observed the GFP-producing cancer cells transporting via the PVS. Results of another study, by Islam et al [57], using a lymphoma xenograft showed that the cells in the cancer PN were human-origin, i.e., from the cancer, not from the host. These cells exhibited stem-cellspecific transcription factor KLF4 at a significantly upregulated level. It is still not clear whether the cancer PVS is
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
8 formed from the host, tumor, or both, and whether these cells are in fact cancer stem cells, all of which are very important information for the cancer metastasis and the sources of cancer stem cells. The study using a GFP-expressing host with nofluorescence-expressing cancer cells [55] may not have complete information on the hostexenograft communication because GFP can provide information from the host only. By the same reason, the study using a GFP-expressing cancer cell line in nonfluorescing animal models was not able to tell us the roles of the host animal in cancer primogenesis. A more complete method suggested here is combining the animal model expressing one fluorescing protein (e.g., GFP) and the cancer cells expressing another fluorescing protein (e.g., red fluorescent protein). This method can help in finding the origin (i.e., the host or the cancer) of the PVS and its subunits by individual fluorescence. A study of the cancer PVS growth path and pattern, and cancer P-microcell production by the color of fluorescence should tell us the role of the PVS in cancer primogenesis and the source of the cancer stem cells.
7. Nontechnical issues In addition to the numerous technical issues that need to be overcome, there are also nontechnical issues that affect the advancement in the PVS science. The PVS is still very new to most scientists, and therefore, the concept that there is another vascular system in the mammalian body has still not been accepted easily. Publishing PVS research results in reputable Western journals and/or receiving research grants for PVS study has also been extremely difficult. In addition, the PVS research community, which is still relatively small, suffers from poor information exchange, which may lead research teams to overlap efforts and funds, slowing the advancement in science. With the current situation, the PVS science is desired to advance in a highly organized, multidisciplinary manner, because the scientific subjects to tackle for the PVS is so broad and extensive. If the PVS community forms a consortium, each PVS research team is in charge of a particular topic of the PVS science, and then shares the study results openly within the community, the advances in the PVS science would be faster. In addition, PVS scientists should attempt to vigorously disseminate the research results via high-quality journal publications, research grant proposals, conference presentations, and lectures, and educate junior PVS scientists. As the PVS science is still new, individuals who recently became involved in the PVS study may knowingly mislead other scientists for their own sake. For example, some scientists have renamed the system (we already have 2 sets of names for the system) and some have avoided revealing important details of the experimental methods in the publications. The potential chaos generated by this type of actions may disorient the scientific community, especially those who desire to participate in PVS research. It could also impede the recognition of validity of the results and terminologies that have already been accepted by the international community. The efforts of Drs Bonghan Kim and Kwang-Sup Soh for establishing the bases of the BHS/PVS science should be respected and honored.
K.A. Kang et al.
8. Conclusion For the past decade, there have been numerous scientific reports and articles on the BHS/PVS. Based on the already reported findings, the PVS has been proven to be an independent vascular system and it may have important roles in mammalian physiology, even though its existence is still not known to many scientists. In this article, we have discussed difficulties to be overcome in the current PVS research approach with suggested new methods. It is our hope that this article will be helpful for PVS scientists in general, and that other scientists would join us to share their ideas to resolve other problems so that we can together elucidate the roles and functions of the PVS in mammals. Ultimately, we hope that accumulated knowledge on the PVS, via effective communication among PVS scientists, is utilized for the prevention, diagnosis, and treatment of diseases.
Disclosure statement The authors have no financial interests related to the material in this manuscript.
References [1] Kim BH. Great Discovery in Biology and MedicinedSubstances of Kyungrak. Pyong Yang, DPRK: Foreign Language Publishing House; 1962. [2] Kim BH. On the Kyungrak System. Pyong Yang, DPRK: The Kyungrak Institute; 1964. [3] Kim BH. Kyungrak System. Proceedings of the Academic of Kyungrak of DPRK, 1965. Pyong Yang, DPRK: Medical Science Press; 1965:9e67. [4] Kim BH. Theory of Sanal. Proceedings of the Academic of Kyungrak of DPRK, 1965. Pyong Yang, DPRK: Medical Science Press; 1965:69e104. [5] Kyungrak Institute of P.D.R. of Korea. Blood cells: Bonghan sanal-circle of cells. J Acad Med Sci. 1965;12:1e7 [In Korean]. [6] Kang KA. A half century gap between Bonghan system and primo vascular system. J Acupunct Meridian Stud. 2013;6: 285e292. [7] Soh KS, Kang KA, Harrison DH. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012. [8] Lim CJ, Lee SY, Ryu PD. Identification of primo-vascular system in abdominal subcutaneous tissue layer of rats. Evid Based Complement Alternat Med. 2015:751937. [9] Yoo JS, Ayati MH, Kim HB, Zhang W, Soh KS. Characterization of the primo-vascular system in the abdominal cavity of the lung cancer mouse model and its differences from the lymphatic system. PLoS ONE. 2010;5:e9940. [10] Ogay V, Soh KS. Identification and characterization of small stem-like cells in the primo vascular system of adult animals. In: Soh KS, Kang KA, Harrison DH, eds. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012:149e156. [11] Hwang S, Lee SJ, Park SH, Chitteti BR, Srour EF, Cooper S, et al. Nonmarrow hematopoiesis occurs in a hyaluronic-acidrich node and duct system in mice. Stem Cells Dev. 2014;23: 2661e2671. [12] Lee SJ, Park SH, Kim YI, Hwang S, Kwon PM, Han IS, et al. Adult stem cells from the hyaluronic acid-rich node and duct system differentiate into neuronal cells and repair brain injury. Stem Cells Dev. 2014;23:2831e2840.
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions [13] Kwon BS, Ha CM, Yu S, Lee BC, Ro JY, Hwang S. Microscopic nodes and ducts inside lymphatics and on the surface of internal organs are rich in granulocytes and secretory granules. Cytokine. 2012;60:587e592. [14] Yoo JS, Kim HB, Won N, Bang J, Kim S, Ahn S, et al. Evidence for an additional metastatic route: in vivo imaging of cancer cells in the primo-vascular system around tumors and organs. Mol Imaging Biol. 2011;13:471e480. [15] Cassidy PJ, Rada GK. Molecular imaging perspectives. J R Soc Interface. 2005;2:133e144. [16] Ciobanu L, Seeber DA, Pennington CH. 3D MR microscopy with resolution 3.7 mm by 3.3 mm by 3.3 mm. J Magn Reson. 2002; 158:178e182. [17] Degena CL, Poggioa M, Mamina HJ, Rettnera CT, Rugara D. Nanoscale magnetic resonance imaging. PNAS. 2009;106: 1313e1317. [18] Nichol JM, Naibert TR, Hemesath ER, Lauhon LJ, Budakian R. Nanoscale Fourier-transform magnetic resonance imaging. Phys Rev X. 2013;3:031016. [19] Lee BC, Yoo JS, Ogay V, Kim KW, Dobberstein H, Soh KS, et al. Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs. Microsc Res Tech. 2007;70: 34e43. [20] Sung B, Kim MS, Lee BC, Yoo JS, Lee SH, Kim YJ, et al. Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs. Naturwissenschaften. 2008;95:117e124. [21] Kim MS, Oh SW, Lim JH, Han SW. Phase contrast x-ray microscopy study of rabbit primo vessels. Appl Phys Lett. 2010;97: 213703. [22] Ogay V, Bae KH, Kim KW, Soh KS. Comparison of the characteristic features of Bonghan ducts, blood and lymphatic capillaries. J Acupunct Meridian Stud. 2009;2:107e117. [23] Lee BC, Kim KW, Soh KS. Visualizing the network of Bonghan ducts in the omentum and peritoneum by using Trypan blue. J Acupunct Meridian Stud. 2009;2:66e70. [24] Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015;111. A3.B.1e3. [25] Jung SJ, Lee SH, Bae KH, Kwon HM, Song YR, Soh KS. Visualization of the primo vascular system, a putative cancer metastasis thread afloat in a lymph duct. Protocol Exchange. 2013. http://dx.doi.org/10.1038/protex.2013.083. [26] Hayat MA. Stains and Cytochemical Methods. New York: Plenum Press; 1993:80e89. [27] Schwartzberg AM, Olson TY, Talley CE, Zhang JZ. Synthesis, characterization, and tunable optical properties of hollow gold nanospheres. J Phys Chem. 2006;110:19935e19944. [28] Carlson E, Perez-Abadia G, Adams S, Zhang JZ, Kang KA, Maldonado C. A novel technique for visualizing intralymphatic primo vascular system using hollow gold nanospheres. J Acupunct Meridian Stud. 2015;8:294e300. [29] Darras JC, de Vernejoul P, Albrarede P. Nuclear medicine and acupuncture: a study on the migration of radioactive tracers after injection at acupoints. Am J Acupunct. 1992;10: 243e256. [30] Darras JC, Albarede P, de Vernejoul P. Nuclear medicine investigation of transmission of acupuncture information. Acupunct Med. 1993;11:22e28. [31] Souza DE, Rebello BM, Silva RC, Agostinho RT, Bastos SRC, Bernardo-Filho M. Nuclear medicine methods in the assessment of acupuncture effects: a short review. Braz Arch Biol Technol. 2007;50:105e110. [32] de Vernejoul P, Albarede P, Darras JC. Nuclear medicine and acupuncture message transmission. J Nucl Med. 1992;33: 409e412. [33] Zhang WB, Tian YY, Li H, Tian JH, Luo MF, Xu FL, et al. A discovery of low hydraulic resistance channel along meridians. J Acupunct Meridian Stud. 2008;1:20e28.
9 [34] Ohnuma K, Yomo T, Asashima M, Kaneko K. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining. BMC Cell Biol. 2006;7:25. [35] Herzenberg NA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA. The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem. 2002;48:1819e1827. [36] Park ES, Lee JH, Kim WJ, Heo J, Shin DM, Leem CH. Expression of stem cell markers in primo vessel of rat. Evid Based Complement Alternat Med. 2013;2013:438079. [37] Gossett DR, Weaver WM, Mach AJ, Hur SC, Tse HT, Lee W, et al. Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem. 2010;397:3249e3267. [38] Fu AY, Spence C, Scherer A, Arnold FH, Quake SR. A microfabricated fluorescence-activated cell sorter. Nat Biotechnol. 1999;17:1109e1111. [39] Soh KS, Kang KA, Ryu YH. 50 years of Bong-Han theory and 10 years of primo vascular system. Evid Based Complement Alternat Med. 2013;2013:587827. [40] Hell SW, Dyba M, Jakobs S. Concepts for nanoscale resolution in fluorescence microscopy. Curr Opin Neurobiol. 2004;14: 599e609. [41] Cremer C, Masters BR. Resolution enhancement techniques in microscopy. Eur Phys J H. 2013;38:281e344. [42] Vainrub A, Pustovyy O, Vodyanoy V. Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser. Optics Lett. 2006;31:2855e2857. [43] Vodyanoy V. High resolution light microscopy of live cells. Microsc Today. 2005;13:26e28. [44] Vodyanoy V, Pustovyy O, Vainrub A. High resolution light microscopy of nanoforms. In: Hoover RB, Levin GV, Rozanov AY, Davies PCW, eds. Instruments, Methods, and Missions for Astrobiology X. Bellingham, WA: SPIE; 2017:1e12. [45] Moore T, Sorokulova I, Pustovyy O, Globa L, Vodyanoy V. Microscopic evaluation of vesicles shed by rat erythrocytes at elevated temperatures. J Therm Biol. 2013;38:487e492. [46] Vodyanoy VJ. Characterization of primo-nodes and vessels by high resolution light microscopy. In: Soh KS, Kang KA, Harrison DH, eds. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012: 83e94. [47] Darzynkiewicz Z. Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. In: Zbigniew D, Harry AC, eds. Methods in Cell Biology. New York: Academic Press; 1990:285e298. [48] Williams RM, Webb WW. Single granule pH cycling in antigeninduced mast cell secretion. J Cell Sci. 2001;113:3839e3850. [49] Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, et al. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 2005;25: 6199e6210. [50] Zhu Z, Xu R. Morphometric observation on the mast cells under the acupuncture meridian lines. Zhen Ci Yan Jiu. 1990; 15:157e158. [51] Zhang D, Ding GH, Shen XY, Yao W, Zhang ZY, Zhang YQ, et al. Role of mast cells in acupuncture effect: a pilot study. Explore. 2008;4:170e177. [52] Lee HS, Lee JY, Kang DI, Kim SH, Lee I, Park SH, et al. Evidence for the primo vascular system above the epicardia of rat hearts. Evid Based Complement Alternat Med. 2013;1e8: 510461. [53] Lim CJ, Yoo JH, Kim Y, Lee SY, Ryu PD. Gross morphological features of the organ surface primo-vascular system revealed by Hemacolor staining. Evid Based Complement Alternat Med. 2013;1e12:350815. [54] Lee VC, Sung B, Eom KH, Park ES, Kim MS, Kim SH, et al. Novel threadlike structures on the surfaces of mammalian abdominal organs are loose bundles of fibrous stroma with
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
10 microchannels embedded with fibroblasts and inflammatory cells. Connect Tissue Res. 2013;54:94e100. [55] Heo C, Hong MY, Jo A, Lee YH, Suh M. Study of the primo vascular system utilizing a melanoma tumor model in a green fluorescence protein expressing mouse. J Acupunct Meridian Stud. 2011;4:198e202. [56] Kang KA, Maldonado C, Perez-Aradia G, An P, Soh KS. Primo vascular system and its role in cancer. Adv Exp Med Biol. 2013; 789:289e296.
K.A. Kang et al. [57] Islam MA, Thomas SD, Sedoris KJ, Slone SP, Alatassi H, Miller DM. Tumor-associated primo vascular system is derived from xenograft, not host. Exp Mol Pathol. 2013;94: 84e90. [58] Akers W, Liu Y, Sudlow G, Lee J, Yoo JS, Lee BC, et al. Identification of primo vascular system in murine tumors and viscera. In: Soh KS, Kang KA, Harrison DH, eds. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012:179e184.
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
MODEL
J Acupunct Meridian Stud 2016;--(-):--e--
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
REVIEW ARTICLE
Technical Challenges in Current Primo Vascular System Research and Potential Solutions Kyung A. Kang 1,*, Claudio Maldonado 2, Vitaly Vodyanoy 3,4 1
Department of Chemical Engineering, University of Louisville, Louisville, KY, USA Department of Physiology and Biophysics, University of Louisville, Louisville, KY, USA 3 Department Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, and School of Kinesiology, Auburn University, Auburn, AL, USA 4 The Research Department, The Edward Via College of Osteopathic Medicine, Auburn, AL, USA 2
Available online - - -
Received: Sep 19, 2015 Revised: Jan 28, 2016 Accepted: Feb 8, 2016 KEYWORDS Bonghan system; primo vascular system; PVS identification; PVS harvest; optical characterization
Abstract Since Bonghan Kim’s discovery of the Bonghan system (BHS) in the 1960s, numerous reports have suggested that the system is fundamental for maintaining mammalian life. The BHS is a circulatory system independent of the blood or the lymphatic system, forms an extensive network throughout the entire mammalian body, has been reported to be the acupuncture meridian, stores distinct types of stem cells, and appears to have some roles in cancer metastasis. Despite Kim’s first report having been published as early as 1962, research progress has been rather slow mainly because the system is very small and translucent, making it optically difficult to distinguish it from the hemoglobin-rich surrounding tissues. Unfortunately, Kim did not describe in detail the methods that he used for identifying and harvesting the system and the components of the system. In 2000, Kwang-Sup Soh reopened the BHS research, and since then, new and important scientific findings on the system have been reported, and many of Kim’s results have been verified. In 2010, the BHS was renamed the primo vascular system. Nevertheless, good tools to properly deal with this system are still lacking. In this article, we address some of the technical difficulties involved in studying the primo vascular system and attempt to discuss potential ways to overcome those difficulties.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. * Corresponding author. Department of Chemical Engineering, University of Louisville, Louisville, KY 40292, USA. E-mail: [email protected] (K.A. Kang). pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2016.02.001 Copyright ª 2016, Medical Association of Pharmacopuncture Institute. Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
2
K.A. Kang et al.
1. Introduction In 1962, Bonghan Kim [1], a scientist in North Korea, reported a discovery of a new vascular system in mammals, which was soon named as the Bonghan system (BHS). The small size (< 3 mm) and optical transparency of the system were probably the main reasons for its delayed discovery. Kim and his research team [2e5] published four additional reports by the end of 1965, and the content of the reports strongly implied the system’s importance in the mammalian physiology. Kim’s claim of the system being the acupuncture meridian particularly surprised the scientific community. Unfortunately, Kim’s research activities suddenly stopped in 1965 [6] and had undergone a dormant period until KwangSup Soh reopened the research on the system in 2000. By 2002, new scientific results on the system started to appear in various journals, and in 2010, the system was renamed as the primo vascular system (PVS) [6,7]. The BHS/PVS consists of the ducts (vessels), corpuscles (nodes), various fluid-containing cells, and biochemicals that flow throughout the system. The following are the terms used during the Kim’s time and those after the Bonghan system became the PVS, placed side by side: Bonghan system Bonghan duct Bonghan ductule Bonghan corpuscle Sanal (or Bonghan sanal) Bonghan liquor
Primo vascular system (PVS) Primo vessel (PV) Subprimo vessel (Sub-PV) Primo node (PN) Primo microcell (P-microcell) Primo fluid (P-fluid)
Kim also named the subunits of sanal as the sanalosome, sanaloplasm, and sanal membrane, which were not renamed by PVS scientists. A new term “primogenesis” was created in 2012, by the University of Louisville team (Louisville, KY, USA), to define the phenomenon of generating or forming the PVS, while describing the new PVS formation on/in the cancer xenografts. Kim [2,3] classified the system into several subtypes by their locations. As his research progressed and more details on the system were uncovered, his classification became also updated. The subtypes are as follows. (1) Internal (or intravascular): floating inside blood and lymphatic vessels (LVs). (2) External (or extravascular): outside the blood vessels and nerves, or independently. (3) Intraexternal: freely on the surface of internal organs in thoracic/ abdominal cavities. (4) Intraorganic: inside organ walls. (5) Neural: in/on the central and peripheral nervous systems. (6) Superficial: in the corium or subcutaneous tissue. This subtype was to be surrounded by dense blood vessels and was close to nerves. Kim reported the corpuscles of this subtype to be acupoints and the ducts to be the acupuncture meridian. Currently, all subtypes except the external subtype are positively identified [7,8]. After observing the path of a radioactive tracer injected into predetermined Bonghan corpuscles, Kim [2,3] concluded that the subtypes closely communicated with each other and that the system did not appear to be controlled by a single central unit, but by multicompartmentalized units.
As Kim’s publications were reports (not journal publications requiring peer review processes), his findings were to be verified before they were taken as scientific facts. Until now, only some of Kim’s findings have been reexamined and verified, and many are still to be investigated. Since 2000, PVS scientists have also reported new, important findings about the system, and some of the notable scientific findings on the BHS/PVS are as follows. (1) Kim claimed that the superficial BHS was the acupuncture meridian [1e5], and, recently, the Ryu team [8] in Korea colocalized the acupoints and superficial Bonghan corpuscles/PNs in the abdomen of animal models, validating Kim’s claim. (2) After 2000, PVS scientists confirmed it to be independent of the blood or lymphatic system by the differences in microanatomical structures and biomarkers [9]. (3) The BHS/PVS is an extensive network throughout the entire mammalian body [3,7]. (4) The Pmicrocells, which Kim named as sanals (live eggs, in Korean), are now confirmed to be unique types of stem cells, and positively confirmed to be performing nonmarrow hematopoiesis and repairing damaged cells/tissues [5,10e12]. (5) Bonghan liquor/P-fluid contains various biochemicals and cells, particularly those associated with immune responses [2,3,13]. (6) The PVS appears to have a role in the dynamics of cancer metastasis [9,14], which is a new finding after 2000. Although only limited facts on the BHS/PVS have been revealed so far, the nature of these listed above clearly demonstrates the importance of the system in the mammalian physiology. Despite the urgency for a thorough investigation on this important system, there are still serious impeding issues in performing PVS research. The most serious one is lacking scientific tools to properly handle its extremely small size (PV, 20e50 mm; PN < 1500 mm) and optical transparency [1e5], which cause difficulties in identifying and harvesting the system. Kim presented his study results logically with reasonable details, but did not describe the methods for identifying and harvesting the PVS in animal models at a level that future scientists can repeat his studies. New techniques for identifying/harvesting the PVS have been reported since 2000, but they are still not satisfactory for most of the scientists, particularly those who are new to the system, to be able to reliably access the PVS sample to perform in-depth investigation. In this article, in an effort to support the scientific advancement in the system, the authors have analyzed important issues to overcome and attempted to suggest potential techniques to resolve these issues. We hope that other PVS scientists also share their ideas with the PVS community. It should be noted that, the term “PVS scientists” in this article is used to represent the scientists who have been involved in PVS research after 2000. For the scientific terms associated with the system, PVS (not BHS) terms are mainly used to avoid unnecessary confusion.
2. Identifying PVS with nanoparticles For properly performing any scientific research, the target sample needs to be identified first. Frequently, utilizing
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions biomarkers specific for the target sample is helpful, but identifying/developing PVS biomarkers has not been realized yet because of the difficulties in obtaining sufficient amount of PVS samples. Identifying the PVS in the entire body, with the help of appropriate contrast agents, using medical imaging modalities could be a way of obtaining sufficient samples. However, the usual spatial resolution obtainable with these tools would be 100 mm at best [15], which is much bigger than the average PV diameter. Although some advanced modalities such as specially designed magnetic resonance imaging [16e18] and electron or X-ray microscopy [19e21] provide exceptionally good resolution, they are not readily available to most scientists and not practical for real-time sample harvesting from animal models. Therefore, the main technique for identifying the PVS has been optical (stereo) microscopy, particularly for studies involving surgeries in live animals, which is the current situation for most PVS studies. Identification of the PVS has been performed by taking advantage of its natural anatomical structure, and the PV is known to have at least three unique characteristics (Fig. 1), according to Kim [2,3] and later PVS scientists [19e22]: (1) multiple sub-PVs in a single PV (Figs. 1A and 1B), and the inter-sub-PV space filled with fibrous, amorphous substances [3]; (2) rod-shaped nuclei of sub-PV endothelial cells (Fig. 1A) [3]; and (3) pores in the PV external wall (Fig. 1B). The second property is helpful for confirming whether the harvested sample is a PV or not. The first and, particularly, the third properties have been effectively utilized for placing appropriate optical contrast agents into the PV. It is still not confirmed whether these pores exist in the outer membrane of all six PVS subtypes, but this property has been used for identifying the PVS inside the LV and the intraexternal PVS [22e24]. Electron microscopy and X-ray microscopy [19e21] have determined the pore size of the outer wall of the rabbit intraexternal PV to be
3 1e5 mm, and the size of the PV or the pore does not seem to vary significantly among the species studied. According to Kim [3], the interstitial space of sub-PVs (ductules) has a fibrous structure and is filled with amorphous substances. Therefore, if particles of an appropriate size (i.e., smaller than the pore in the PV wall, but similar to or slightly larger than the spacing in the fibrous structure) are applied to the PV from outside, they are expected to get into the PV via the pores and be retained in the interstitial space. If these particles possess an appropriate optical property, then they would generate optical contrast for the PV. Fig. 2 schematically describes the mechanism of identifying the PVS inside an LV (intra-LV PVS) using nano-sized contrast agents. The LV is a good site for identifying the PVS because it has a translucent vessel wall and its fluid is clear, and, therefore, the optically contrasted, intra-LV PVS can be seen through the LV wall. In this procedure, the contrast agent at an appropriate size is first injected into the lymph node connected to the target LV until the LV is filled with the particles (Fig. 2A), and in time, the particles would enter the PV via the pores. The natural flow of lymphatic fluid clears particles from the LV (outside the PV), while the particles that have already entered the PV remain in the interstitial space, providing optical contrast to the PV (Fig. 2B). One such method exploits the unstable nature of Alcian blue at a pH of > 5.6 [25]. Alcian blue is dark green in color, its molecular size is 2e3 nm, and it is frequently used in staining acidic mono- or polysaccharides of biological samples in histological studies [26]. This dye itself, however, does not appear to stain the PVS preferentially to the LV. For the purpose of PVS identification, Alcian blue powder is first dissolved in PBS (pH 7.4); the solution is boiled (possibly to maximize its solubility in PBS) and filtered to remove large particles. The Alcian blue solution is then injected into the lymph node of the animal until the
Figure 1 Properties unique for the PV. (A) PV illustrated in Kim’s third report (slightly modified from [3]; 1-sub-PV; 2- sub-PV endothelial cell; 3-rod shaped nucleus of endothelial cell; 4-PV external layer; 5-nucleus of PV endothelial cell. (B) Schematic diagram of a PV cross section: pores in the PV outermost wall; multiple sub-PVs in a PV; and fibrous nature of PV interstitial space. PV Z primo vessel; sub-PV Z subprimo vessel. Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
4
K.A. Kang et al.
A
B
Sub-PV
Lymphatic vessel
Nanocon trast agent
Pore on PV wall
PV Ly ymphatic flluid flow Figure 2 Schematic diagrams illustrating the identification of the PVS inside an LV using nano-sized contrast agent. (A) PVScontaining LV is filled with nanoparticles, by injecting them into the lymph node. The particles get into the PV via the pores of the PV external vessel wall. (B) The LV is cleared by its natural fluid flow, while the PVS retains the particles trapped in it, generating optical contrast. LV Z lymphatic vessel; PV Z primo vessel; PVS Z primo vascular system; sub-PV Z subprimo vessel.
LV is filled with it [25]. During the process, the dye forms small particles due to its instability in the PBS and lymphatic fluid at pH 7.4. The actual size of the formed Alcian blue particles is not known, but they go through the pore in the PV outer membrane and remain in the interstitial space, generating contrast for the PVS. The dye preparation and injection steps should to be carried out within a short period of time (not clearly defined yet) before larger particle formation and/or precipitation starts. A more controlled and user-friendly method is using nano-sized particles of an appropriate size range and optical contrast against hemoglobin. The University of Louisville team has developed a technique utilizing hollow gold nanoparticles (HGNs) with a color range from green to blue [27]. HGNs were selected because gold is biocompatible and the size and color of HGNs can be controlled well. In a preliminary study using the rat model, particles in the size range of 50e125 nm worked well, with a success rate of 95% [28]. This method has advantages over the Alcian blue method, because the HGNs have a relatively homogeneous size distribution, preparation steps are simpler, and the PVS in the LV becomes visible within 20 minutes after the particle injection. The nanoparticle optical contrast agent used for the purpose of intra-LV PVS identification requires certain properties when used in live animals: (1) it must be nontoxic, biocompatible, and chemically inert because it is to be injected into LVs; (2) the particle surface should be relatively hydrophilic and should not easily aggregate; and (3) it needs to provide a strong optical contrast against the tissue background (i.e., hemoglobin color). According to
our experiments, dark green, greenish blue, or dark blue seems to fit well for the purpose. Up to now, five out of the six PVS subtypes (except the external subtype) have been positively confirmed. The superficial PVS may also need to be given special attention because this subtype is said to be directly related to the acupuncture therapy, and a thorough understanding of this subtype may help in optimizing the therapy. Kim [2] illustrated how the radioactive tracer injected to a Bonghan corpuscle (PN) in the skin moved to other corpuscles via the meridian. Besides the study by Kim, there have been several studies on tracing meridian paths using radioactive tracers or dyes, by injecting them into a particular acupoint (not Bonghan corpuscles or PNs) [29e33]. Since dealing with radioactive tracers is not ideal, the method developed by Lim et al [8] for the colocalization of PNs and acupoints in the abdominal hypodermis region of rats using the threesolution Hemacolor system may be a better approach. The subtype that is most difficult to identify seems to be the external PVS subtype, judging from the lack of scientific articles on this subtype. The external PVS was reported to be covered with connective tissue, according to Kim [3], and, therefore, accessibility to the pores on its external wall is still not known.
3. Harvesting PVS via microelectromechanical system As described in the Introduction, most PVS scientists have already recognized the potential health benefits that may be achieved by appropriately manipulating the nature of
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions the PVS, e.g., regenerating tissues using one’s own Pmicrocells or detecting cancer via the abnormally formed PVS, etc. However, much of the fundamental information about the PVS (e.g., micro- and submicroanatomical structures, biological compositions, biomarkers, physiological functions, the entire PVS map in the body, and communication between the PVS and other organs) still remains to be studied before any translational work can be done. Besides, we still need to verify all of Kim’s findings, which were published 50 years ago. To acquire these basic scientific information, PVS samples need to be available in a reliable manner, but acquiring a sufficient amount of PVS samples has been difficult because of its small size as well as its identification-related issues already described. The sites that may potentially provide a relatively large amount of PVS samples are the blood and LVs because the PVS runs inside them and they are long. Out of the two, LVs are better candidates, as mentioned earlier. Identification of the intra-LV PVS can be performed more easily in live animals because the floating motion of the PVS in the lymphatic fluid helps identify it better. Once the LV loses its fluid and becomes flattened, the PVS easily sticks to the LV inner wall. Live animals, however, breathe and the target LV also moves with the breathing motion, which makes the harvesting tasks more challenging. Therefore, keeping the target LV still during the breathing motion may be helpful (e.g., fixing the LV on a solid surface). Next, retrieving the PVS from the LV requires extremely delicate microsurgical techniques with good microsized tools. A few medical tools, such as micro-extra-fine surgical knives and grippers (e.g., Excelta, Buellton, CA, USA; nanoScience Instruments, Phoenix, AZ, USA), are currently available for this type of study, although some of them can be expensive and may still need to be tailored for PVS research. The tip of the atomic force microscope may potentially serve as a microknife. A suggested method for the intra-LV PVS, once it is identified, is as follows: make small incisions at two positions on the LV, depending upon the desired amount of PVS samples; then insert the tips of two sets of microgrippers (e.g., nanoScience Instruments) into the two respective incision sites of the LV, to hold the two ends of the PVS; cut the LV along its length between the two incision sites; and cut the two ends of the PVS (outer sides of the grippers) and retrieve the freed PVS by lifting the grippers. One might suggest to first harvest the PVS-containing LV and then retrieve the PVS from it, in order to simplify the process. However, when the LV is cut without holding the two ends of the PVS, the PVS tends to curl up inside the LV because of its elastic nature. Fig. 3 shows the PVS optically contrasted by HGNs, before (Fig. 3A) and after (Fig. 3B) the LV-containing PVS was cut at its lower part, and the PVS is shown to curl up after the LV was cut. In addition, if PVS retrieval is not performed immediately after the LV is cut, then the PVS may stick to the inner wall of the LV, which makes it difficult to harvest the PVS. Since the entire procedure involved in the PVS harvest requires precise operation due to the small target sample size, if the procedure is performed by a micromanipulator based on the microelectromechanical system (MEMS) technology, human errors may be minimized. It is also
5
Figure 3 A PVS identified inside an LV, using HGNs. (A) The PVS contrasted by the HGNs can be seen inside the LV. (B) After the lower end of the PVS bearing LV is cut, the PVS is curled up in the LV. HGN Z hollow gold nanosphere; LV Z lymphatic vessel; PN Z primo node; PV Z primo vessel; PVS Z primo vascular system.
highly desirable to have microliter-sized P-fluid collectors and/or microinjectors, utilizing MEMS technology, for the P-fluid analysis and applying biochemicals into the PVS.
4. P-microcell retrieval by cell sorting techniques Kim [4] reported that there are unique, self-regenerating cells (P-microcells/sanals) inside the PVS, particularly in the PNs [5], and these cells are now positively confirmed to be stem cells [11,12]. A thorough understanding of Pmicrocells and their roles would, therefore, be very important for the stem cell science and regenerative medicine, and retrieving these cells from PNs in a reliable manner is the first step toward it. This may be done, first, by mechanically or enzymatically disintegrating the outer layer of PNs to obtain all cells inside them, and then separating the P-microcells from other cells. Since the constituents or properties of the PN membrane are still not clearly known, development of an optimized process for removing the membranes may have to wait until they are well characterized. After the membranes are removed, P-microcells need to be separated. Over the past decade, cell-sorting techniques have been advanced significantly because detecting circulating cancer cells has been emphasized as an important research topic for early cancer diagnosis. The Pmicrocell is characterized by its small size, 1e3 mm
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
6 [10e12], while most cells are in the range of 10 mm. Therefore, P-microcells may be separated from the cell population in PNs by flow cytometry, either using the difference in cell size [34] or by fluorescence [35] after labeling the cells with fluorophores via antibodies against stem cell biomarkers that P-microcells express [10,36]. For PVS studies, it may also be advantageous to use micro-sized cell-sorting systems [37,38] instead of using a regular flow cytometer, since PNs are very small, and the cell volume and number in a PN are very small.
5. Optical microscopy for PVS characterization Optical microscopy was one of the most important methods used in the discovery of the BHS in the 1960s, and rediscovery of the PVS in the 2000s [39], although it was used mainly for PVS identification instead of characterization. If it is to be used for PVS characterization, the micro- and/or submicrostructural components and translucent optical property of the PVS, however, present serious challenges. In the visible light transmission and reflection microscopy, even for advanced confocal devices, the lateral image resolution would not exceed 180 nm [40], requiring substantially enhanced resolution [41] for detailed structural characterization of the PVS. Optical resolution up to the submicron level may, however, be achieved via the annular illumination to increase the intensity of diffraction fringes, and via the coherent illumination of small (compared to wavelength) objects to
K.A. Kang et al. allow the image edge to become sharper, thus increasing the resolution [42e46]. Minimizing the light spot size on the sample and reducing the stray light can also improve spatial resolution and contrast: reducing the spot dimension raises the irradiance of the sample and increases the contrast by increasing the interaction with small particles in the sample. The photon density of the system modified by the Auburn University team (Auburn, AL, USA) became approximately 150 times higher than that of the conventional Nikon darkfield condenser (Eclipse E400, Oil 1.43-1.20, Japan). This increased illumination intensity raises the optical difference between tiny light-scattering particles and their background. By doing so, all particles larger than 90 nm, including small DNA granules and P-microcells, can be resolved [42]. A high-output numerical aperture of an illumination system allows utilizing high numerical apertures of the microscope objective and produces highly luminous super-resolution images. Figs. 4 and 5 are images with significantly enhanced resolution and contrast, taken by the Auburn University team with proper adjustments described above. Fig. 4 shows an image of an intraexternal PN connected to a PV [3], which was harvested from the surface of the rat intestine. PNs are usually linked to the PVs at their both ends, and in some cases, several PVs are connected to a single PN. This particular PN was, however, connected to the PV at its one end only (Figs. 4A and 4B). Figs. 4B and 4D are enlarged images of the rectangular sections in Figs. 4A and 4C, respectively. The PVS sample was treated with acridine
Figure 4 Optical images of a nonfixed, intraexternal PVS from a rat, stained with acridine orange. (A) Overall view of the PVS. (B) A magnified image of the part connecting the PN and PV in Fig. 4A. (C) A magnified image of the branched PV, shown in the dotted rectangle in Fig. 4A. (D) A further magnified view of the PV shown in the dotted rectangle in Fig. 4C. Its cell nuclei are shown with arrows. The scale bars in A, B, C, and D are 500 mm, 200 mm, 100 mm, and 50 mm, respectively. PN Z primo node; PV Z primo vessel; PVS Z primo vascular system. Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions
7
Figure 5 A transverse sectional view of a small PN. (A) In the brightfield mode, basophilic granules are labeled with black stars. (B) The same section photographed in the darkfield mode. Vacuoles are indicated by white arrows, intravascular space by a white arrow head, cluster of granules by a black arrow, a granule in the small lumen by a black arrow head, the largest lumen by a white star, and the interrupted portion of lumen connected to a vacuole by a dotted white arrow. C Z capsule; L Z lumens; PN Z primo node.
orange, which is used as a nucleic-acid-selective fluorescent cationic dye. The staining reveals cells fluorescing with green and red light: the light at a wavelength of 502 nm excites the acridine orange/DNA complex that emits at 525 nm (green). When it interacts with RNA, excitation shifts to 460 nm and emission to 650 nm (red) [47]. In these images, the details of this PVS may be very clearly seen: the PN has an oval shape with dimensions of 0.85 1.1 mm2 and the PV length is 1.25 mm. The diameter of the middle part of the vessel is w140 mm. The apical end of the vessel is branched into two smaller vessels. The branched vessels are connected to other nodes (not shown here). The green and red colors may be interpreted as DNA and RNA of cell nuclei, but acridine orange also accumulates and emits red light in mast secretory granules and other cellular acidic compartments [48,49]. In Fig. 4D, the red color is emitted from the cell cytoplasm rather than from the cell nuclei. The nuclei of these cells (shown by arrows) are green. These particular cells represent w5% of the entire cell population. Microscopic observation of the mast cells under the acupuncture meridian lines of humans and rats revealed that the mast cells were more concentrated under the meridian lines in comparison with their control areas. Therefore, the red-stained cells in the figure may be mast cells [50e53]. According to this result, the mast cells represent w5% of acridine orange-stained cells in the PVS, which is close to 4%, as quantified by Lim et al [53], but less than 20%, as reported by Kwon et al [13]. The difference may be due to variations in techniques and experimental conditions. Fig. 5 illustrates the transversal section of a PN in the bright and dark field modes (Figs. 5A and 5B, respectively). The walls of sub-PVs are smooth, single layered, and uninterrupted, except for the wall of the lumen labeled by the white star. There is a small opening in the wall that leads to a vacuole (dotted white arrow). Some sub-PVs run very close to each other, with a small intravascular space between them (white arrow head). The section also shows randomly scattered single cells and granules, and some granules are in clusters (black arrow). These features shown in the optical image of the PN cross section agree with those described by electron and light microscopy in general [22,54].
As illustrated here, optical microscopy with proper adjustment can be utilized effectively for PVS characterization in a micron/submicron level.
6. Studying cancer primogenesis via fluorescent protein Most PVS studies performed up to now have focused on the identification and basic characterization of the system probably because the system is still new. However, there have been interesting preliminary study results on the behaviors of the PVS in/adjacent to cancerous tumors (cancer PVS), at least for six different cancer xenografts. Although this topic is not associated with basic characterizations of the normal PVS, we included it in the article because cancer affects many people, and a thorough understanding of the cancer PVS may lead to better management of the disease. The cancer xenografts studied up to now are: lung (NCI-H460) and ovarian (SK-OV-3) cancer [9,14], cutaneous melanoma (B16 F10) [55], breast cancer (MDA-MB231), gastric cancer (MKN12) [56], human leukemia (U937) [57], and also human breast cancer mimicking cell line 4T1 [58]. The PVS in/on normal tissues or organs is usually neither very densely populated nor easily observed. The cancer PVS, however, has been shown to form at a higher density in/around the tumor. In addition, cancer PVS has been shown to participate in cancer cell transport from the primary to the secondary tumors [9,14]. To learn how the cancer PVS proliferates, Heo et al [55] used a combination of green fluorescent protein (GFP)expressing host animals and nonfluorescing human skin cancer cell line, and the result showed that GFP PVS was formed inside the tumor. The Soh team [56] used a GFPexpressing cancer cell line in nonfluorescing animal models and observed the GFP-producing cancer cells transporting via the PVS. Results of another study, by Islam et al [57], using a lymphoma xenograft showed that the cells in the cancer PN were human-origin, i.e., from the cancer, not from the host. These cells exhibited stem-cellspecific transcription factor KLF4 at a significantly upregulated level. It is still not clear whether the cancer PVS is
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
8 formed from the host, tumor, or both, and whether these cells are in fact cancer stem cells, all of which are very important information for the cancer metastasis and the sources of cancer stem cells. The study using a GFP-expressing host with nofluorescence-expressing cancer cells [55] may not have complete information on the hostexenograft communication because GFP can provide information from the host only. By the same reason, the study using a GFP-expressing cancer cell line in nonfluorescing animal models was not able to tell us the roles of the host animal in cancer primogenesis. A more complete method suggested here is combining the animal model expressing one fluorescing protein (e.g., GFP) and the cancer cells expressing another fluorescing protein (e.g., red fluorescent protein). This method can help in finding the origin (i.e., the host or the cancer) of the PVS and its subunits by individual fluorescence. A study of the cancer PVS growth path and pattern, and cancer P-microcell production by the color of fluorescence should tell us the role of the PVS in cancer primogenesis and the source of the cancer stem cells.
7. Nontechnical issues In addition to the numerous technical issues that need to be overcome, there are also nontechnical issues that affect the advancement in the PVS science. The PVS is still very new to most scientists, and therefore, the concept that there is another vascular system in the mammalian body has still not been accepted easily. Publishing PVS research results in reputable Western journals and/or receiving research grants for PVS study has also been extremely difficult. In addition, the PVS research community, which is still relatively small, suffers from poor information exchange, which may lead research teams to overlap efforts and funds, slowing the advancement in science. With the current situation, the PVS science is desired to advance in a highly organized, multidisciplinary manner, because the scientific subjects to tackle for the PVS is so broad and extensive. If the PVS community forms a consortium, each PVS research team is in charge of a particular topic of the PVS science, and then shares the study results openly within the community, the advances in the PVS science would be faster. In addition, PVS scientists should attempt to vigorously disseminate the research results via high-quality journal publications, research grant proposals, conference presentations, and lectures, and educate junior PVS scientists. As the PVS science is still new, individuals who recently became involved in the PVS study may knowingly mislead other scientists for their own sake. For example, some scientists have renamed the system (we already have 2 sets of names for the system) and some have avoided revealing important details of the experimental methods in the publications. The potential chaos generated by this type of actions may disorient the scientific community, especially those who desire to participate in PVS research. It could also impede the recognition of validity of the results and terminologies that have already been accepted by the international community. The efforts of Drs Bonghan Kim and Kwang-Sup Soh for establishing the bases of the BHS/PVS science should be respected and honored.
K.A. Kang et al.
8. Conclusion For the past decade, there have been numerous scientific reports and articles on the BHS/PVS. Based on the already reported findings, the PVS has been proven to be an independent vascular system and it may have important roles in mammalian physiology, even though its existence is still not known to many scientists. In this article, we have discussed difficulties to be overcome in the current PVS research approach with suggested new methods. It is our hope that this article will be helpful for PVS scientists in general, and that other scientists would join us to share their ideas to resolve other problems so that we can together elucidate the roles and functions of the PVS in mammals. Ultimately, we hope that accumulated knowledge on the PVS, via effective communication among PVS scientists, is utilized for the prevention, diagnosis, and treatment of diseases.
Disclosure statement The authors have no financial interests related to the material in this manuscript.
References [1] Kim BH. Great Discovery in Biology and MedicinedSubstances of Kyungrak. Pyong Yang, DPRK: Foreign Language Publishing House; 1962. [2] Kim BH. On the Kyungrak System. Pyong Yang, DPRK: The Kyungrak Institute; 1964. [3] Kim BH. Kyungrak System. Proceedings of the Academic of Kyungrak of DPRK, 1965. Pyong Yang, DPRK: Medical Science Press; 1965:9e67. [4] Kim BH. Theory of Sanal. Proceedings of the Academic of Kyungrak of DPRK, 1965. Pyong Yang, DPRK: Medical Science Press; 1965:69e104. [5] Kyungrak Institute of P.D.R. of Korea. Blood cells: Bonghan sanal-circle of cells. J Acad Med Sci. 1965;12:1e7 [In Korean]. [6] Kang KA. A half century gap between Bonghan system and primo vascular system. J Acupunct Meridian Stud. 2013;6: 285e292. [7] Soh KS, Kang KA, Harrison DH. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012. [8] Lim CJ, Lee SY, Ryu PD. Identification of primo-vascular system in abdominal subcutaneous tissue layer of rats. Evid Based Complement Alternat Med. 2015:751937. [9] Yoo JS, Ayati MH, Kim HB, Zhang W, Soh KS. Characterization of the primo-vascular system in the abdominal cavity of the lung cancer mouse model and its differences from the lymphatic system. PLoS ONE. 2010;5:e9940. [10] Ogay V, Soh KS. Identification and characterization of small stem-like cells in the primo vascular system of adult animals. In: Soh KS, Kang KA, Harrison DH, eds. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012:149e156. [11] Hwang S, Lee SJ, Park SH, Chitteti BR, Srour EF, Cooper S, et al. Nonmarrow hematopoiesis occurs in a hyaluronic-acidrich node and duct system in mice. Stem Cells Dev. 2014;23: 2661e2671. [12] Lee SJ, Park SH, Kim YI, Hwang S, Kwon PM, Han IS, et al. Adult stem cells from the hyaluronic acid-rich node and duct system differentiate into neuronal cells and repair brain injury. Stem Cells Dev. 2014;23:2831e2840.
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
Technical Challenges in PVS Research and Solutions [13] Kwon BS, Ha CM, Yu S, Lee BC, Ro JY, Hwang S. Microscopic nodes and ducts inside lymphatics and on the surface of internal organs are rich in granulocytes and secretory granules. Cytokine. 2012;60:587e592. [14] Yoo JS, Kim HB, Won N, Bang J, Kim S, Ahn S, et al. Evidence for an additional metastatic route: in vivo imaging of cancer cells in the primo-vascular system around tumors and organs. Mol Imaging Biol. 2011;13:471e480. [15] Cassidy PJ, Rada GK. Molecular imaging perspectives. J R Soc Interface. 2005;2:133e144. [16] Ciobanu L, Seeber DA, Pennington CH. 3D MR microscopy with resolution 3.7 mm by 3.3 mm by 3.3 mm. J Magn Reson. 2002; 158:178e182. [17] Degena CL, Poggioa M, Mamina HJ, Rettnera CT, Rugara D. Nanoscale magnetic resonance imaging. PNAS. 2009;106: 1313e1317. [18] Nichol JM, Naibert TR, Hemesath ER, Lauhon LJ, Budakian R. Nanoscale Fourier-transform magnetic resonance imaging. Phys Rev X. 2013;3:031016. [19] Lee BC, Yoo JS, Ogay V, Kim KW, Dobberstein H, Soh KS, et al. Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs. Microsc Res Tech. 2007;70: 34e43. [20] Sung B, Kim MS, Lee BC, Yoo JS, Lee SH, Kim YJ, et al. Measurement of flow speed in the channels of novel threadlike structures on the surfaces of mammalian organs. Naturwissenschaften. 2008;95:117e124. [21] Kim MS, Oh SW, Lim JH, Han SW. Phase contrast x-ray microscopy study of rabbit primo vessels. Appl Phys Lett. 2010;97: 213703. [22] Ogay V, Bae KH, Kim KW, Soh KS. Comparison of the characteristic features of Bonghan ducts, blood and lymphatic capillaries. J Acupunct Meridian Stud. 2009;2:107e117. [23] Lee BC, Kim KW, Soh KS. Visualizing the network of Bonghan ducts in the omentum and peritoneum by using Trypan blue. J Acupunct Meridian Stud. 2009;2:66e70. [24] Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015;111. A3.B.1e3. [25] Jung SJ, Lee SH, Bae KH, Kwon HM, Song YR, Soh KS. Visualization of the primo vascular system, a putative cancer metastasis thread afloat in a lymph duct. Protocol Exchange. 2013. http://dx.doi.org/10.1038/protex.2013.083. [26] Hayat MA. Stains and Cytochemical Methods. New York: Plenum Press; 1993:80e89. [27] Schwartzberg AM, Olson TY, Talley CE, Zhang JZ. Synthesis, characterization, and tunable optical properties of hollow gold nanospheres. J Phys Chem. 2006;110:19935e19944. [28] Carlson E, Perez-Abadia G, Adams S, Zhang JZ, Kang KA, Maldonado C. A novel technique for visualizing intralymphatic primo vascular system using hollow gold nanospheres. J Acupunct Meridian Stud. 2015;8:294e300. [29] Darras JC, de Vernejoul P, Albrarede P. Nuclear medicine and acupuncture: a study on the migration of radioactive tracers after injection at acupoints. Am J Acupunct. 1992;10: 243e256. [30] Darras JC, Albarede P, de Vernejoul P. Nuclear medicine investigation of transmission of acupuncture information. Acupunct Med. 1993;11:22e28. [31] Souza DE, Rebello BM, Silva RC, Agostinho RT, Bastos SRC, Bernardo-Filho M. Nuclear medicine methods in the assessment of acupuncture effects: a short review. Braz Arch Biol Technol. 2007;50:105e110. [32] de Vernejoul P, Albarede P, Darras JC. Nuclear medicine and acupuncture message transmission. J Nucl Med. 1992;33: 409e412. [33] Zhang WB, Tian YY, Li H, Tian JH, Luo MF, Xu FL, et al. A discovery of low hydraulic resistance channel along meridians. J Acupunct Meridian Stud. 2008;1:20e28.
9 [34] Ohnuma K, Yomo T, Asashima M, Kaneko K. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining. BMC Cell Biol. 2006;7:25. [35] Herzenberg NA, Parks D, Sahaf B, Perez O, Roederer M, Herzenberg LA. The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem. 2002;48:1819e1827. [36] Park ES, Lee JH, Kim WJ, Heo J, Shin DM, Leem CH. Expression of stem cell markers in primo vessel of rat. Evid Based Complement Alternat Med. 2013;2013:438079. [37] Gossett DR, Weaver WM, Mach AJ, Hur SC, Tse HT, Lee W, et al. Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem. 2010;397:3249e3267. [38] Fu AY, Spence C, Scherer A, Arnold FH, Quake SR. A microfabricated fluorescence-activated cell sorter. Nat Biotechnol. 1999;17:1109e1111. [39] Soh KS, Kang KA, Ryu YH. 50 years of Bong-Han theory and 10 years of primo vascular system. Evid Based Complement Alternat Med. 2013;2013:587827. [40] Hell SW, Dyba M, Jakobs S. Concepts for nanoscale resolution in fluorescence microscopy. Curr Opin Neurobiol. 2004;14: 599e609. [41] Cremer C, Masters BR. Resolution enhancement techniques in microscopy. Eur Phys J H. 2013;38:281e344. [42] Vainrub A, Pustovyy O, Vodyanoy V. Resolution of 90 nm (lambda/5) in an optical transmission microscope with an annular condenser. Optics Lett. 2006;31:2855e2857. [43] Vodyanoy V. High resolution light microscopy of live cells. Microsc Today. 2005;13:26e28. [44] Vodyanoy V, Pustovyy O, Vainrub A. High resolution light microscopy of nanoforms. In: Hoover RB, Levin GV, Rozanov AY, Davies PCW, eds. Instruments, Methods, and Missions for Astrobiology X. Bellingham, WA: SPIE; 2017:1e12. [45] Moore T, Sorokulova I, Pustovyy O, Globa L, Vodyanoy V. Microscopic evaluation of vesicles shed by rat erythrocytes at elevated temperatures. J Therm Biol. 2013;38:487e492. [46] Vodyanoy VJ. Characterization of primo-nodes and vessels by high resolution light microscopy. In: Soh KS, Kang KA, Harrison DH, eds. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012: 83e94. [47] Darzynkiewicz Z. Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. In: Zbigniew D, Harry AC, eds. Methods in Cell Biology. New York: Academic Press; 1990:285e298. [48] Williams RM, Webb WW. Single granule pH cycling in antigeninduced mast cell secretion. J Cell Sci. 2001;113:3839e3850. [49] Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, et al. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 2005;25: 6199e6210. [50] Zhu Z, Xu R. Morphometric observation on the mast cells under the acupuncture meridian lines. Zhen Ci Yan Jiu. 1990; 15:157e158. [51] Zhang D, Ding GH, Shen XY, Yao W, Zhang ZY, Zhang YQ, et al. Role of mast cells in acupuncture effect: a pilot study. Explore. 2008;4:170e177. [52] Lee HS, Lee JY, Kang DI, Kim SH, Lee I, Park SH, et al. Evidence for the primo vascular system above the epicardia of rat hearts. Evid Based Complement Alternat Med. 2013;1e8: 510461. [53] Lim CJ, Yoo JH, Kim Y, Lee SY, Ryu PD. Gross morphological features of the organ surface primo-vascular system revealed by Hemacolor staining. Evid Based Complement Alternat Med. 2013;1e12:350815. [54] Lee VC, Sung B, Eom KH, Park ES, Kim MS, Kim SH, et al. Novel threadlike structures on the surfaces of mammalian abdominal organs are loose bundles of fibrous stroma with
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001
+
MODEL
10 microchannels embedded with fibroblasts and inflammatory cells. Connect Tissue Res. 2013;54:94e100. [55] Heo C, Hong MY, Jo A, Lee YH, Suh M. Study of the primo vascular system utilizing a melanoma tumor model in a green fluorescence protein expressing mouse. J Acupunct Meridian Stud. 2011;4:198e202. [56] Kang KA, Maldonado C, Perez-Aradia G, An P, Soh KS. Primo vascular system and its role in cancer. Adv Exp Med Biol. 2013; 789:289e296.
K.A. Kang et al. [57] Islam MA, Thomas SD, Sedoris KJ, Slone SP, Alatassi H, Miller DM. Tumor-associated primo vascular system is derived from xenograft, not host. Exp Mol Pathol. 2013;94: 84e90. [58] Akers W, Liu Y, Sudlow G, Lee J, Yoo JS, Lee BC, et al. Identification of primo vascular system in murine tumors and viscera. In: Soh KS, Kang KA, Harrison DH, eds. The Primo Vascular System: Its Role in Cancer and Regeneration. New York: Springer; 2012:179e184.
Please cite this article in press as: Kang KA, et al., Technical Challenges in Current Primo Vascular System Research and Potential Solutions, Journal of Acupuncture and Meridian Studies (2016), http://dx.doi.org/10.1016/j.jams.2016.02.001