Numerous studies have reported the existence of an additional vascular system in mammals: the primo vascular system, consisting of primo vessels and primo nodes. We investigated lymphatic vessels from rats by staining lymphatic tissue with the dye Alcian Blue to identify whether they contain primo vessels. In one exceptional specimen, we were able to clearly identify a primo vessel inside a lymphatic vessel. Microscopy images of this specimen are shown in this report and analyzed. Our report is intended to document our findings and to motivate others to repeat and extend our study in order to investigate in detail the presence and physiological role of primo vessels in lymphatic vessels.

Comprising lymphatic vessels (LVs), lymph nodes (LNs), lymphoid organs (such as the thymus and spleen), and lymphatic fluid, the lymphatic systems serve important physiological functions in organisms including immunological surveillance, fluid homeostasis (interstitial fluid removal from the tissue) as well as absorption and transport of fats and fatty acids^[1][2][3]. Although the lymphatic system is well studied, novel anatomical and physiological aspects of it are still being discovered, such as the ability of the autonomic nervous system to control lymphatic vessels^[4], the existence of LVs in the meninges^[5][6][7], the fundamental role of the lymphatic system for renal physiology and pathology^[8] or the ability of LVs to transition into blood vessels in adult microvascular networks during microvascular remodeling^[9].

For several decades, researchers have claimed to have discovered a vascular system distinct from the blood and lymph vascular system. The initial discovery by the North Korean researcher Bong-Han Kim^[10][11] was rediscovered decades later by the South Korean research group of Kwang-Sup Soh. Since then, the phenomenon has been continuously investigated but is still almost unknown by Western scientists^[12][13].

Soh termed the novel anatomical structure “primo vascular system” (PVS) comprising “primo vessels” (PVs) and “primo nodes” (PNs)^[13]. Based on immunochemistry, histology, and genetic analysis, several studies have demonstrated so far that the PVS is distinct from blood vessels, lymphatic vessels, or nerves^{[14][15][16][17][18][19][20][21]}. The PVS has been detected in several animals (e.g. dogs, rats, mice) and in human tissue^[13]. In terms of morphology, the PVs of the PVS are about 20–150 µm in width, are filled with a liquid^[22], and comprise subvessels and sinuses (i.e. tubular structures inside the PVs and PNs)^[23]. PVs are difficult to observe in vivo since they are semitransparent. The dyes Trypan Blue and Alcian Blue have been found to help in identifying the PVS in vivo due to the dyes’ strong ability to stain the PVS. Research so far has identified several physiological functions of the PVS, including its role as a niche and possible transport route for stem cells or stem cell-like cells^{[24][25][26][27][28]}, immune function^[29], tissue regeneration^[25][26], erythropoiesis^[30][31][32] as well as in transporting microvesicles and exosomes^[33][34].

PVs have been detected in several different anatomical locations in organisms, including on the surface of organs^{[16][35][23][20][36][37][22]}, inside and along blood vessels^[38][39][40], and, fascinatingly, inside LVs^{[41][10][42][43][40][44][45][14][46][47]}.

We have previously reported on the microscopy analysis of the PVS of rats and our discovery of a red threadlike structure inside PVs and PNs taken from the intestine surface (possibly indicating extramedullary hematopoiesis occurring inside the PVS)^[32]. The aim of the present publication is to document a well-preserved PVS specimen that shows a PV within an LV in a level of detail never before published.

From the LVs extracted from the vena cava of a rat, one LV (with a length of about 9.5 mm) was found to clearly contain a PV (Fig. 1). As figure 1(A-D) shows, the PV is clearly visible as a blue thread-like structure due to the Alcian Blue staining. The microscopic images show the ultrastructure of the PV traversing the LV: (i) The PV lies inside the LV, appearing coiled and circular, possibly due to the rupture of the PV (indicated by an asterisk in Fig.1(A)) and a subsequent shrinking of it. (ii) The PV is about one-third the size of the LV (diameter of the LV: about 150 µm, the diameter of the PV: about 50 µm). (iii) At the anterior part of the LV, a part of the PV can be seen in a coiled form, seemingly the result of a rupture of the whole PV. The tissue in vivo is unlikely to be curled or coiled. (iv) The PV seems to be free-floating within the LV and is not attached to the inner lumen of the LV.

Our result confirming the presence of a PV inside an LV is in agreement with previously published results documenting a PV inside an LV^{[41][10][42][43][40][44][45][14][46][47]}. The first observation that LVs can have PVs inside was made by Kim in the 1960s^[10], but no photographic evidence was provided in that report, nor was there a description of how to detect PVs in lymphatic specimens. It was Lee et al.^[40] in 2005 that presented for the first time microscopic images of LVs with a PV inside (stained with Janus Green B) (diameter of the LVs: 786±5, a diameter of the PV: 154±1 µm, ratio: 5:1). 13 samples of LVs with a PV inside were found in this study and analyzed. The tissue was extracted from rabbits. In this study, the authors provided in particular microscopic evidence of a PN passing through the valve of an LV. In a follow-up study, Lee et al.^[44] presented 6 additional specimens extracted from rabbit tissue showing a PV inside an LV (diameter of the LVs: 970±3, a diameter of the PV: 53±2 µm, ratio: 18:1). Johng et al.^[41] were the first to analyze the tissue of rats and found specimens of LVs with a PV inside (diameter of the LVs: 240±7, a diameter of the PV: 52±3 µm, ratio: 4.5:1). In agreement with the findings of Lee et al.^[40], this study also documented an LV with a PN inside that passes through an LV valve. The study used cobalt-ferrite magnetic nanoparticles to stain the PVS tissue. Staining with these nanoparticles was also made in the study by Yoo et al.^[46] where also LVs with a PV inside were documented. The study was done with rats. In a study by Lee and Soh^[43], the authors also reported the detection of LVs from rabbits with a PV inside (diameter of the LVs: 519±139, a diameter of the PV: 32±8 µm, ratio: 16:1). In this study, an LV was found that clearly showed a PV inside that was exiting the LV wall at one point (see Fig. 5B in their publication), proving that the PV can permeate (i.e. enter or exit) an LV. The authors also reported that they were able to pull out a PV from inside an LV, showing that the PV is not tightly attached to the LV wall inside. Lee et al.^[45] reported the discovery of an LV from mouse tumor tissue with a PV inside. Shin et al.^[14] analyzed LVs from rabbits that contain PVs and found statistically significant differences in gene expression from both tissues, proving that the PV tissue is not identical to LV. An innovative method to study PVs inside LVs in vivo in rats was developed by Kim et al.^[42]. The authors develop a window chamber system attached to the skin that allows long-term monitoring of PVs inside LVs along the superficial epigastric vessels.

Given the significance of this finding for our understanding of anatomy, it is important to ask why such a novel secondary vessel from the PVS has not been observed by many more researchers investigating tissue of the lymphatic system. We also question why the existence of the PVS is not already well known by the scientific community and documented in anatomical textbooks. We believe there are two main reasons for this. First, PVs are semitransparent and easily overlooked when investigating tissue in general and LVs in particular. Second, although several papers have been published about the existence of PVS tissue inside lymphatics, these reports are not well known and have so far, unfortunately, not attracted a great amount of attention from the scientific community.

The authors of the present report would welcome a detailed and objective study of the PVS without any bias. The existence of the PVS in general, and the existence of PVs inside LVs, should be validated and investigated independently and critically by as many research institutions as possible worldwide. A critical step for the successful validation of our findings is first to find PVs in or on the surface of the tissue. There are two protocols published so far^[48][49] that help to guide through the procedure to find PVs. Furthermore, the authors of the present manuscript volunteer also to train interested researchers directly in finding the PVS.

In this report, we presented an exceptionally clear example of an LV with a PV inside. Our finding is in agreement with other published studies reporting the existence of a secondary vessel, different from an LV, that can be found inside an LV. We agree with the conclusion of Yoo et al. that the “mere demonstration of the existence of this novel structure inside lymphatic vessels is a remarkable event in current anatomy and heralds a breakthrough toward establishing a new anatomical system completely different from the blood vascular, the lymphatic, and the neural systems”^[46]. Further studies should replicate and extend our observation with a systematic analysis of the LVs of organisms and the possible role of PVs inside. Despite the anatomical analysis, a detailed analysis is required to understand the physiological significance of the PVS as well as the PVs inside LVs in particular.

Our study has two main limitations: First, only one specimen showing an LV with a PV inside is presented. The reason for this is mainly that the detection of such a clear and characteristic specimen as shown in this report is difficult and seldom. Second, only optical microscopy has been used in our study. Additional investigation with immunohistochemistry (for example using monoclonal antibodies specific to the PVS, as recently shown^[50][51]), along classical dyes for lymphatic tissue, endothelial cells, stem cells, etc.), electron microscopy, and X-ray microcomputed tomography (as recently shown to be a valuable tool for PVS characterization^[52] would have been advantageous and future studies should perform such analysis.

For this study, one male Sprague-Dawley rat (Orient Bio, Gyeonggi-do, Korea; age: 6 weeks) was used. The rat was housed with other rats in a temperature-controlled room (20–26°C) under a 12 h light/dark cycle with food and water available ad libitum. The rat was not prepared specifically. A young rat was chosen since young rats have less fat attached to the surface of their intestine which simplifies the detection of the PVS tissue.

Alcian Blue solution (1%) was made by 0.05 g (Sigma, St. Louis, MO, USA) and dissolved in 15 ml phosphate-buffered saline (PBS, pH 7.4), then filtered through a 0.2 mm filter with a 10 ml syringe.

After the rat was anesthetized by an intramuscular injection of an anesthetic cocktail (alfaxalone, 41.7 mg/kg, intraperitoneally; xylazine. 16 mg/kg, intraperitoneally), it was placed under a stereomicroscope (OSM-1, Dongwon, Seoul, Korea) and the abdomen was opened. The surgical opening of the abdomen was carefully conducted so as not to cut blood vessels and to stop minimal bleedings immediately to avoid the blood entering the abdominal cavity.

Alcian Blue solution (1%, 0.2–0.3 ml) was injected into the lymphatic node nearby the lumbar (lumbar node) which being explored without any solution leaking from the node. After 3–5 min, the lymphatic vessel was isolated for further analysis. Why the dyes Trypan Blue and Alcian Blue have a strong ability to stain the PVS is currently an empirical finding that is not completely understood. It is known, however, that Alcian blue (a cationic dye) stains hyaluronic acid (an anionic, nonsulfated glycosaminoglycan) by binding to anionic residues of hyaluronic acids; since the PVS contains hyaluronic acid, this aspect is possibly relevant to explain the ability of the dye to stain the PVS^[53][54]. Trypan blue likely stains in particular the pores, sinuses or gaps on the PVS tissue surface, and seems to be washed away from the PVS more slowly compared to other tissue^[55].

The sample was analyzed under a phase-contrast microscope (IX 70 inverted microscope; Olympus Optical Co., LTD, Tokyo, Japan) and images were taken. Image analysis was performed with ImageJ 1.52a and figure 1 was created using Adobe Photoshop (CS6) and Adobe Illustrator (CS6). The raw microscopic images of the specimen shown in figure 1 (n = 14) were stitched together. The white balance of the images was corrected and the background removed.

The research project was enabled by the 2018 SNF Scientific Exchange grant (no. 180409) to F.S. and the National Research Foundation of Korea grant (2018R1D1A1B07043448) to P.D.R.

The author would like to thank Rachel Scholkmann for proofreading the manuscript.

The authors declare no conflicts of interest.

The experimental protocols involving animals were approved by the local Ethics Committee.

The animal experiments performed were in accordance with the guidelines of the Laboratory Animal Care Advisory Committee of Seoul National University and were approved by the Institute of Laboratory Animal Resource of Seoul National University (SNU-140926-2).

No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.