Primo Bundles Identified by Microcomputed Tomography in Primo Vascular Tissue on the Surface of Rat Abdominal Organs

Abstract

Background

The primo vascular system (PVS) is a novel network composed of primo nodes (PNs) and primo vessels (PVs). Currently, its anatomy is not fully understood.

Objectives

The aim of this study was to elucidate the three-dimensional PN–PV structure.

Methods

Organ-surface PVS tissue was isolated from healthy and anemic rats. The tissues were analyzed by X-ray microcomputed tomography (CT), hematoxylin and eosin staining, and scanning electron microscopy.

Results

From CT images, we identified one or more bundles in a PV. In the PN, the bundles were enlarged and existed in isolation and/or in anastomosis. The transverse CT images revealed four areas of distinct intensities: zero, low, intermediate, and high. The first two were considered to be the sinuses and the subvessels of the PVS and were identified in the hematoxylin and eosin–stained PN sections. The enlargement of the PN from anemic rats was associated with an increase in the intermediate-intensity area. The high-intensity area demarcated the bundle and was overlapped with the mesothelial cells. In scanning electron microscopy, the PV bundles branched out, tapering down to a single bundle at some distance from the PN. Each bundle was composed of several subvessels (~5 µm). Clustered round microcells (1–25 µm), scattered flat oval cells (~15 µm), and amorphous extracellular matrix were observed on the surface of the PVS tissue.

Conclusions

The results newly showed that the primo bundle is a structural unit of both PVs and PNs. A bundle was demarcated by high CT intensity and mesothelial cells and consisted of multiple subvessels. The PN bundles contained also sinuses.

1. Introduction

The primo vascular system (PVS) was originally reported as an anatomical entity of the acupuncture meridian by Kim [1, 2] in the early 1960s and was rediscovered by Soh [3] in the early 2000s (see the study by Soh [3] for review). The current name was adopted by the organizing committee of the first international symposium on the system in 2010 [4]. The PVS is a whole-body network composed of nodes and vessels that connecting nodes to other nodes and to target cells. In this network, primo fluid circulates together with primo microcells containing DNA molecules. Acupuncture stimulation induces an electrical signal and movement of hormones and microcells within the PVs (see the study by Vodyanoy et al [5] for a reintroduction of Kim’s work).

In the last 20 years, many of Kim’s findings on the structure and function of the PVS have been confirmed. PVS tissues have been identified in most parts of the body, including the surface of the abdominal organs, the interior of the blood and lymphatic vessels, the central nervous system, and the internal organs [3].

As established in light microscopic studies, the primo vessels (PVs) of the organ-surface PVS (osPVS) tissue are not single tubes, but bundles of subvessels [1, 6–8]. The PVs give off smaller branches and taper with distance from the node, and their fine terminals seem to make delicate contact with tissues including fat cells [9, 10].

A primo node (PN) is not a single sinus, but seems to preserve the PV structures inside [11, 12]. Numerous small round cells are found in PNs [1, 8, 13, 14], and the outer area of PN sections shows higher cellularity [8]. Recently, Lim et al. [15] reported the presence of mesothelial cells on the surface of subunits of the PV and the PN of osPVS tissue. The location and morphology of mesothelial cells are very similar to those of squamous epithelial cells with flattened nuclei reported previously [16]. Small pores or openings of 0.2–3 µm were identified on and between these cells [15–18]. It is also known that the PNs of the osPVS tissue are enlarged more than twofold in rats with heart failure or hemolytic anemia [19–21].

PVS tissue has been also reported to function as a circulatory channel by Kim [1], which was also confirmed by Han et al [16] and Sung et al [22]. The flow rate of the dye (Alcian blue) through rabbit osPVS tissue was 0.3 m/s through the PVs [22]. Alcian blue injected into the BL23 acupoint was found selectively in the osPVS tissues at 2 hours after injection [6]. The dye used in these studies is likely to flow through the subvessels composing the PVs that were initially reported by Kim [1] and confirmed by Shin et al. [9], Vodyanoy [7], and Lim et al. [8]. A unique feature of the subvessels inside PNs is their reticulated interlacement and basophilic granules inside the subvessels [1]. Similar features have also been reported by Lim et al [8]. They found a honeycomb-like reticular pattern of the sub-vessels (~10 µm) on Hemacolor-stained slices (200 µm) of fresh PNs from osPVS tissue. Basophilic granules (1–2 µm) are found inside and along the border of these subvessels.

Although previous research on the structure of PVS tissues has expanded our knowledge, integrative images of the entire structure of the osPVS tissue are not yet available. Most studies were based on images of thin tissue slices and or tissues heavily processed for microscopic studies. X-ray microcomputed tomography (micro-CT) is an effective tool to obtain quantitative three-dimensional (3D) virtual images of small nonmineralized biological samples such as the insect brain and mouse embryo [23]. One can get a series of transverse or horizontal section images of a whole tissue without making actual tissue sections. Using micro-CT, Lee et al. [24] showed that there are two channels inside the PV in intralymphatic PVS tissue based on two-dimensional (2D) images. Detailed 3D virtual section images of the whole PVS tissue that allow visualization of the inner structures have not yet been reported. In this study, we analyzed the 3D structure of osPVS tissue using micro-CT and further confirmed the micro-CT findings by light and electron microscopy. We chose the rat osPVS tissue because it has been most frequently studied than other PVS tissues.

2. Materials and methods

2.1. Animal preparations

Male Sprague Dawley rats (5 weeks; Orient Bio, Gyeonggi-do, Korea) were used and kept in an atmosphere at a controlled temperature (20–26°C) and humidity (40–70%) under a 12-h:12-h light–dark cycle. All the experiments were conducted in accordance with the Guide for the Laboratory Animal Care Advisory Committee of Seoul National University and approved by the Institute of Laboratory Animal Resources of Seoul National University (SNU-180827-1). A rat model of hemolytic anemia was prepared by administering phenylhydrazine for 2 consecutive days (40 mg/kg, intraperitoneal injections), and acute anemia was confirmed as reported previously [21]. Under deep anesthesia, the rat abdomen was incised along the midline, and the PVS (milky colored semitransparent tissue consisting of nodes and vessels) was sampled from the surface of internal organs under a stereo-microscope, as reported previously [8].

Immediately before mounting the PVS tissue on the CT scanner, the tissue was removed from the solution, and excess solution was gently soaked out using a tissue paper. The tissue was then positioned between two pieces of Styrofoam supports (approximately 20 × 5 × 2 mm), and the supports were completely wrapped with parafilm to prevent them from drying out during scanning. The tissue was mounted on the stage of a micro-CT scanner (SkyScan 1172; Bruker, Kontich, Belgium). The isolated tissue was fixed overnight in 2% paraformaldehyde at 40 kV and 200 µA with a pixel size of 0.95 µm (Figs. 1–4 except Fig. 2B) or 1.9 µm (Fig. 2B), a rotation angle of 180°, a rotation step of 0.3°, and an exposure time of 1176 ms. The total scan time was about 2 hours. The projection images of 600–2000 sections were reconstructed using CTAn (version 1.16; Bruker, Kontich, Belgium) software. The Skyscan softwares (DataViewer and CTVox; Bruker, Kontich, Belgium) used to analyze the 2D (cross-sectional and longitudinal sections) and 3D CT images, respectively. In the present study, we used Styrofoam supports to maintain a linear orientation of the tissue, which is observed in abdominal organs [8]. The CT contrast signals that originated from the Styrofoam were weak, were scattered around the PVS tissue, and did not overlap with the tissue signals (Fig. 3 and Supplementary Fig. 1). When constructing the 3D images, these artifacts were removed manually [Fig. 1 and Supplementary Video 1 (0.95-µm pixel) and Video 2 (1.9-µm pixel)]

Figure 1

Surface structure of rat primo vascular tissue visualized by micro-CT. The three-dimensional micro-CT images representing the primo node (PN; width = 100–243 µm) and its two distal parts, the primo vessels (PVs; width = 45–65 µm) at 0° (A), 90° (B), 180° (C). and 270° (D). Note that the downward PV is composed of multiple thread-like structures (marked by asterisks) and maintains its structure within the PN. Some thread-like structures are detached (marked by a plus sign in A). Note also the slender and long thread-like structures (marked by white arrows), the dark (low intensity) area in the PN that must be an artifact of sample preparation (B and D), and small granules of lower contrast (red arrows, D). The images in A and C are narrower than those in B and C and likely owing to compression of the tissue mounted between two pieces of Styrofoam (see Materials and methods and Supplementary Video 1). CT = computed tomography.

Figure 2

Micro-CT virtual sections of two primo vascular tissue samples from rats. (A) A three-dimensional (3D) surface image (90°) of the primo vascular system (PVS) tissue shown in Fig. 1 (left) and its transverse segments (right) at the primo node (PN) (a2) and primo vessel (PV) (a1 and a3). Virtual 3D segments (thickness = 33 µm) at the levels of a1, a2, and a3 are shown at a 45° forward tilt. Note the vessel-like structures, particularly in a2 and a3.(B) Three-dimensional images (90°) of another PVS tissue sample (left) and its cut surface images at the levels of the PN (b1–b4) and PV (b5–b8) (see Supplementary Video 2). The right panel presents cross-sectional images of the PVS tissue at the levels marked by b1–b8. The insets (b2-1 andb8-1) show the squared areas of the images b2 and b8 in an enlarged form to illustrate the low-contrast circular areas (~10 µm) inside the PN and PV bundles (thread-like structures in Fig. 1). Note also the higher contrast (brighter) boundary of the bundles and the larger low-contrast areas (yellow arrows) inside the bundles. CT = computed tomography.

Figure 3

Distribution of contrast in the color scale of the PVS tissue shown in Fig. 1. (A) Cross-sectional images of a virtual slide of the primo node (PN). The X-ray contrast is presented by a color scale intensity from 0 to 255 arbitrary units (a.u.) as in the inset. Note the four different areas of different intensity: 1 (black; 0 a.u.), 2 (dark scarlet to dark coral; 30–60 a.u.), 3 (green–yellow to light magenta; 98–120 a.u.), and 4 (blue to sky blue; 180–210 a.u.). The scattered background signals are from the Styrofoam support. (B) Intensity distribution along two lines, b1 and b2 in A. The dotted lines of dark coral, green–yellow, cobalt blue, and sky blue colors correspond to intensities of 60, 98, 180, and 210 a.u., respectively. The lowest intensity level is 0, which corresponds to the black area in the axis “a1” or the background. (C) A sagittal image of the tissue shown in A (90°). Note the blue violet to cobalt blue colors (>180 a.u.) at the boundaries and the continuous dark areas (0 a.u.) inside the tissue. (D) Short- and long-axis diameters of lower intensity areas (subvessels) in the PN and primo vessel (PV) measured at 60 a.u. (n =35 and 22 measurements). (E) Distances between subvessels in the PN and PV (n =80 and 55 measurements). (F) Short- and long-axis diameters of the dark areas (sinuses) in the PN and PV measured at 0 a.u. (n =16 and 23 measurements from 4 sections). *p < 0.05; **p < 0.01. PVS =primo vascular system.

Figure 4

Micro-CT images of a primo node (PN) obtained from PVS tissue of an anemic rat. (A) CT images of the PN in an anemic rat. (a1) Surface images in grayscale and cross-sectional images in color scale at low and higher magnifications (a2 and a3). Note that the bundles were not well developed in the PN from the anemic rat. (B) The intensity plot of the color scale images on the long (b1) and short axes (b2) of the cross section at the levels marked by green arrows shown in A (a2). (C) Distance of the low-intensity area measured at the level of 30 a.u. (n = 35 and n = 35 from 7 sections in both the control and anemic rats). ***p < 0.001. The control data were obtained from the tissue presented in Figs. 1–3. (D) Short- and long-axis diameters of sinuses in the PN and primo vessel measured at 0 a.u. (n = 16 and 32 measurements from 4 sections). CT = computed tomography; PVS = primo vascular system.

Supplementary video related to this article can be found at https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jams.2020.07.002

The PVS tissue was initially fixed overnight in 10% neutral buffered formalin, processed, embedded in paraffin, and cross-sectioned at 4 µm. The resulting PVS sections were stained with hematoxylin and eosin (H&E) as part of routine intake procedures. For scanning electron microscopy (SEM), the sampled osPVS tissues were kept in 2% paraformaldehyde for 24 hours, processed further as per the procedures reported previously [25], and observed under field-emission SEM (SIGMA; Carl Zeiss, Oberkochen, Germany). All data are expressed as mean ± standard error. The Student t-test was used to analyze the data. The threshold for statistical significance was set at p <0.05.

3. Results

The surface and inner structures of the osPVS tissue, as imaged by micro-CT, are presented in Figs. 1–4 and a 3D video (Supplementary Video 1 and 2). Fig. 1 shows the surface features of a PN with bipolar PVs at four different angles. In light of the reported width of PNs of the osPVS (340–1500 µm; [8]), this PN is relatively small. In this figure, the PN is wider at 90° (Fig. 1B) and 270° (Fig. 1D) than at 0° (Fig. 1A) and 180° (Fig. 1C), which appears to have resulted from compression by the Styrofoam supports used for micro-CT scanning. The PN appeared as a mixture of several thread-like structures (marked by yellow asterisks) and multiple branches of various sizes. In Fig. 1A, three such structures are clearly visible, and these seemed to form the PN. In Fig. 1B, one thread-like structure was discontinued abruptly (marked by the plus sign), which likely resulted from detachment during tissue preparation. Smaller thread-like structures were also found to run along the PN obliquely (marked by white arrows). Small, dark (low-intensity) particles were also identified on the surface of the images at 90° (Fig. 1B) and 270° (Fig. 1D; marked by red arrows). These surface features can be confirmed in detail in the supplementary 3D video (Supplementary Video 1).

Fig. 2 shows the virtual transverse sections of two primo vascular tissues expressed using gray color levels [0–255 arbitrary units (a.u.)]. The intensity of the CT contrast was scaled from the lowest (black, 0 a.u.) to the highest (white, 255 a.u.). Fig. 2A shows three segments (33 µm) of a PN with two PVs, and one can identify vessel-like structures in the transverse segments of the PV and PN tilted forward at an angle of 45°. The grayscale intensity was higher at the boundaries of the major thread-like structures of the PN and PV. There were many circular areas of lower intensity inside the PN and the PV. These low-intensity areas appeared longitudinally along the PN and PV and hence are considered to be the sinuses of vessels of various sizes (Supplementary Video 1). In the transverse sections, the boundaries of thread-like structures in the PN were clearly identified in isolation, as shown in Fig. 2 (a2). Within the PN, the thread-like structures seemed to maintain their structure and/ or merge together to form a larger thread-like structure and sinuses (0 a.u.). Fig. 2B illustrates the CT images of a medium-sized PN. The surface images of the PN are similar to those in Figs. 1 and 2A. Because we observed abrupt cuts in this tissue, we assumed that it was a PN with multipolar PVs (see Supplementary Video 2). Inside the PN, the thread-like structures were found to be in close contact, and their boundaries were less obvious than those shown in Fig. 2A. A thread-like structure became enlarged and merged with others (Fig. 2B, b1–b4). Within the thread-like structures, circular areas of low intensity (~10 µm in diameter) were found in both PNs and PVs (Fig. 2B, insets); these are considered to be the “subvessels” of the PVS [1, 2, 5]. The large low-intensity areas (marked by yellow arrows) also appear inside the thread-like structures, which are considered the sinus of the PVS [16, 17]. Because the morphology of the “thread-like structure” observed so far fits well in the morphological features of the “bundle” of primo subvessels [1, 2, 5, 7, 9, 17], the thread-like structure will be called the bundle from now on.

In the grayscale images, it was difficult to accurately differentiate the intensity by eye. To characterize the distribution of intensity levels in the CT images more precisely, as shown in Fig. 3A, we transformed the CT contrast of 256 levels into a color scale (black to green). We then plotted the color-coded CT intensity along the axes of the long (a1) and short axes (a2), as shown in Fig. 3B. The distribution and frequency of the following 6 color levels were distinctive: black (0 a.u.), dark coral (60 a.u.), green–yellow (98 a.u.), cobalt blue (180 a.u.), and sky blue (210 a.u.). These intensity levels were expressed as colored lines in the intensity plot (Fig. 3B). The area of lowest intensity (black, “1” in Fig. 3A) had an intensity level of 0 and was likely to be the sinuses of the PVS tissue [16, 17]. The low-intensity areas (30–60 a.u.; “2” in Fig. 3A) were found in all bundles of the PVS, and its inner intensity did not decrease to zero. In the 2D transverse images, a green–yellow belt (98 a.u.; ~1 µm) demarcated the inner intermediate-intensity areas (98–120 a.u.; “3” in Fig. 3A) from the high-intensity boundaries (130–210 a.u.; “4” in Fig. 3A). In summary, we were able to identify four different areas (structures) from the virtual cross sections of the micro-CT images: (1) zero-intensity areas (black; 0 a.u.), (2) low-intensity areas (dark scarlet to dark coral; 30–60 a.u.), (3) intermediate-intensity areas (green–yellow to light magenta; 98–120 a.u.), and (4) high-intensity areas (blue to sky blue, 180–210 a.u.). The low-intensity areas (30–60 a.u.) occupied a higher proportion than the areas of any other intensity (Fig. 3A and B). A similar intensity pattern was observed in longitudinal sections of the primo vascular tissue (Fig. 3C).

Based on the results of the analyses shown in Fig. 3B, we measured the diameters of the low-intensity areas (<60 a.u). The diameters of the short and the long axes were 1.77 ± 0.16 and 3.36 ± 0.43 µm in the PVs (n = 22) and 1.51 ± 0.13 and 2.91 ± 0.31 µm in the PNs (n = 35), respectively. No significant difference in diameter was identified between the PNs and the PVs (p = 0.208 and 0.396, respectively, Fig. 3D). The number of the low-intensity areas was 38, 4, and 9 in the top, middle, and bottom bundles of the PN in Fig. 3A, respectively. We were able to identify 10–19 low-intensity areas in the virtual cross sections of the PV of the PVS tissue shown in Fig.1 (data not illustrated). Based on these observations, we consider the low-intensity areas to be the primo subvessels (Bonghan ductules) [1, 2, 4] because of their location and quantity [1, 2, 5].

In general, the number of the subvessels per unit area was higher in the PVs, and the interval between the nearest subvessels in the PNs was significantly larger than that in the PVs (8.75 ± 0.59 µm, n = 80 vs. 6.93 ± 0.61 m; n = 55, respectively; p = 0.039; Fig. 3E). Assuming that the lowest intensity (0 a.u.; Fig. 3) corresponds to the sinus of PVS tissue [16, 17], when we compared the PNs and the PVs, the short and the long axes of the sinuses in the PNs were significantly longer (4.63 ± 0.44 and 10.19 ± 1.13 µm, n = 16) than those in the PVs (3.35 ± 0.39 and 6.48 ± 0.71 µm, n = 23; p = 0.038 and 0.006, respectively, Fig. 3F).

In previous studies, we observed that the osPVS tissue was enlarged more than two times in rats with heart failure or hemolytic anemia [20, 21]. Fig. 4A illustrates a 3D CT surface image of the PN part in a representative enlarged sample of PVS tissue from an anemic rat. The PN of the anemic rat showed rough surface structures with its major bundles mingled together and small particles (Fig. 4A, a1). Its surface structures were similar to those of the PNs in normal rats except for its size (150–670 µm). In a cross section at the level marked by the arrowhead, the dark areas (sinus; 0 a.u.) in both tissues are easily identified at higher magnification (Fig. 4A, a2 and a3). The boundary of the bundles is not well developed inside the PN, unlike the control rats shown in Figs. 1–3. Interestingly, there is a lower intensity belt (dark coral) that can be barely noticed in the outer area (Fig. 4A, a2). The signals were most frequent in the range of 60–90 a.u. (medium-intensity area between dark coral and green–yellow; Fig. 4B) in the plots of color scale intensities of the long (b1) and short axes (b2). Interestingly, the low-intensity areas (30–60 a.u) appeared less frequent in Fig. 4A (a2). In support of this impression, the distance between the low-intensity areas (measured at 30 a.u.) was significantly larger in anemic than in control rats (239 ± 28 µm, n = 35 vs. 29 ± 2.0 µm, n = 35, respectively; p = 0.001; Fig. 4C). The transverse images of the anemic rat PVs were also filled with intermediate-intensity spots and appeared more compact than those of the PVs from normal rats (not illustrated). No significant differences were identified between the diameters of the short and long axes of the sinuses of the PN and PV in the anemic rats (Fig. 4D), unlike what was found in the control rat PVs (see Fig. 3F). In addition, there was no significant difference in the size of the PN and PV sinuses between the control and anemic groups. The short and long axes of PN sinuses were 4.63 ± 0.44 and 10.19 ± 1.13 µm in the control rats (n = 16) and 5.00 ± 0.47 and 10.00 ± 1.11 µm in the anemic rats (n = 16), respectively (p = 0.5612 and 0.9066 for short and long axes, respectively). The short and long axes of PV sinuses were 3.35 ± 0.39 and 6.48 ± 0.71 µm in the control rats (n = 23) and 4.56 ± 0.43 and 7.84 ± 0.88 µm in the anemic rats (n = 32), respectively (p = 0.0513 for short and 0.2618 for long axes, respectively). Collectively, the results shown in Fig. 4 indicate that the main difference detected in the enlarged PN of anemic rats was an increase in the intermediate-intensity areas.

The analysis of CT contrast in the transverse sections of the PN revealed the presence of vessels and sinuses. We examined the potential vessels and sinuses in thin PN sections stained with H&E (Fig. 5). In general, the PN sections were rich in small round cells with large nuclei and little cytoplasm. In the PN section shown in Fig. 5, some bundles could be identified by the narrow space between the bundles (marked by asterisks), but the boundaries of individual bundles were not readily identified. At higher magnification, the sinuses (large clear spaces of various shapes) appeared heavily collapsed and surrounded by thin nuclei of squamous cells (black arrows), as shown in Fig. 5A–C. The long axis of the largest sinus was ~100 µm. The sinuses were continuously identified at the same location along the bundles in the PN, but were not observed in the PV (not illustrated). In addition, there were small round clear areas of 10–20 µm, which we consider subvessel-like structures. The luminal borders were readily identified in these structures (Fig. 5D–F). At the outer border of the bundle, cells with flattened nuclei were found on the surface (marked by white arrow heads in Fig. 5B), and these cells are considered to be mesothelial cells [15]. Connective tissues and fibrous cells with spindle-shaped nuclei were not identified around these structures in the PN. Round cells, red blood cell–like cells, and other substances were also identified in the sinuses of the bundles and subvessel-like structures (Fig. 5B–F). Collectively, the sinuses and subvessel-like structures in the thin PN section stained with H&E are likely to correspond to the zero-intensity and low-intensity areas in the CT images (see also Fig. 2B). The diameters of the sinuses and subvessel-like structure in the H&E-stained thin sections were much larger than those measured based on the color intensity scale (see Figs. 3 and 4).

Figure 5

Sinuses and subvessels in the primo node (PN) of organ-surface PVS tissue. (A) A PN section (4 µm) stained with hematoxylin and eosin. The boundary is obvious in some bundles (*). (B and C) Representative sinuses are shown at higher magnification. The diameters of the long and short axes of the collapsed sinuses are 100 and 44 µm in A and are 44 and 17 µm in B, respectively. Note the flat mesothelial cells on the PN surface (white arrowheads). (D–F) Representative subvessel-like structures at higher magnifications. (D) The long- and short-axis diameters of these structures are 10 and 9, respectively. (E) The long- and short-axis diameters of these structures are 35 and 24, respectively. (F) The long- and short-axis diameters of these structures are 17 and 14 µm, respectively. Note the membrane-like structures (indicated by black arrows) lining the inner surface of the sinuses and the subvessel-like structures that contain cells and other substances (indicated by gray arrows). PVS = primo vascular system.

To further investigate the surface features of the PVS tissue shown in Figs. 1–4, we examined the rat osPVS tissue by SEM. Fig. 6 presents SEM images of a typical sample of osPVS tissue. In this tissue sample, one can see the PVs only on the right side of the PN. The PN in this tissue sample has two PVs toward both the left and right sides, but the left one is curved back, and runs across the PN body (“b”) and further along the right PV (Fig. 6B). Another smaller branch is curved back to the left (curved arrow). It is unlikely that the PVS tissue is curved and coiled in vivo, as shown in Fig. 6, and this must reflect artifacts that occurred when processing the tissue for SEM analysis (Fig. 1; see also Fig. 1 of the study by Shin et al. [9] and Fig. 3 of the study by Lim et al. [25]). The PV abruptly becomes smaller at the point (asterisk), likely owing to detachment of the bundle. The PV got smaller with distance from the PN, but thicker at some points (see the part between the asterisk and “d”). The PN body does not look like a single tube, and one can identify multiple bundles or branches of various sizes, which resemble the features revealed by micro-CT scans shown in Figs. 1, 2B, and 4A. The proximal part of the PV is composed of multiple bundles measuring 50–100 µm [Fig. 6A (c) and 6C], and the distal or terminal PV is represented by a single PV bundle [Fig. 6A (d) and 6D]. It is of note that the terminal single PV bundle is also composed of finer 5-µm-wide subvessels. On the surface, there are clustered small round cells with a rough surface (5–10 µm), round cells with a smooth surface (1–25 µm), and flat oval cells (~15 µm) scattered over the PN body and PN–PV transition (inset and arrow heads in Fig. 6B). The latter are considered as the mesothelial cells reported in the osPVS tissue previously [15]. Most surfaces were covered by amorphous extracellular matrix [18] that seems to cover cell-like structures (inset of Fig. 6C) and subvessels (Fig. 6D). Dense longitudinal fibers were located in the inner surface of the PV, and loose circular fibers were located in the outer surface of the PV (inset of Fig. 6A). We also found thin circular fibers (200–500 nm) crossing the PV bundles at 90° with an interval of 1–3 µm (inset of Figs. 1 and 6D). In general, the gross features of the PN and PV on SEM are consistent with those of the surface structures of the PN shown by 3D CT in Figs. 1 and 5.

Figure 6

Scanning electron microscopy (SEM) micrographs of PVS tissue isolated from the surface of abdominal organs. (A) SEM micrograph of a whole PVS tissue sample (~4.5 cm) at a low magnification. The primo node (PN), the enlarged, abruptly decreased, and terminal parts of primo vessel (PV) are indicated by “b,” “c,” “*,” and “d,” respectively. The inset illustrates the squared part of the PV at higher magnification. Note longitudinal fibers, circular submicron fibers, and extracellular matrix. (B) The PN and proximal PV marked by “b” are shown at higher magnification. Note the flattened cells scattered (arrow heads), the PV branch curved back to the left (curved arrow), and the other branch underneath running upward and to the right. The inset illustrates the PN–PV transition area marked by a dotted circle at higher magnification. Note also the presence of small cells (~5 µm) on the surface and flat oval cells (~15 µm in length) covering a branch of the PV. (C) A thicker part of the PV marked by “c” in A at higher magnification. Note the presence of three bundles (width: ~100 µm) and small cells on the surface. The inset illustrates the squared area at higher magnification. Note thick amorphous extracellular matrix covering the cell-like structures. (D) A distal part of the right PV marked by “d” at higher magnification. Note several subvessels (width: ~5 µm), submicron fibers crossing the subvessels, and amorphous substances on the surface. The scales for panel A, B, C, and D are 500, 200, 50, and 5 µm and those in the insets of A, B, and C are 2, 5, and 2 µm, respectively. PVS = primo vascular system.

4. Discussion

Micro-CT and microscopy revealed the following morphological features of the rat osPVS tissue. The PV is composed of several bundles. Inside the PN, the PV bundles are enlarged and are present in isolation and/or in anastomosis. The bundles of the PV branch out and become smaller with distance from the PN. A single PV bundle is composed of multiple subvessels (8–19) of ~5 µm and sinuses. Transverse CT images show four different areas of zero, low, intermediate, and high contrast inside the PVS tissue. The zero-intensity and low-intensity areas are likely to be the sinuses and subvessels of the PVS. The high-intensity areas are located on the boundary of the bundles, which is covered by mesothelial cells. The intermediate-intensity areas are observed within the bundles and around the high-intensity boundary. The area of intermediate intensity is increased in the tissue of anemic rats.

Our findings on the gross structure of the organ-surface PN are consistent with previous reports in that the PV structures can be identified within the PN. Kim [1, 2, 5] described the PN as a structure formed by the ramifications and anastomoses of subvessels. Yi et al. [11] reported that the PV maintains its boundary and curls around after entering the PN. Vodyanoy [7] reported that the PN is heterogeneous in structure and filled with tightly spun capillary bundles without an external capsule. In the present study, the PV structures within the PN ran along the longitudinal axis of the PV–PN–PV path (see Supplementary Video 1 and 2), unlike previous reports [7, 11]. The PVs within the PN maintain their boundaries in isolation, but merge with the nearby PVs at other points, and their diameters are variable (Figs. 1–3). This observation is consistent with that of Scholkmann et al. [12], who showed a continuous red line through three PNs in a path containing four PNs. Because we scanned the whole tissue by micro-CT, the natural morphology is likely to have been better conserved during the experimental procedures.

4.1. Primo bundles

The gross morphology of a typical PV has been reported to involve a single vessel covered by a periductium (external jacket) with 1–20 subvessels (ductules) of 5–15 µm in diameter [1, 2, 5]. Interestingly, in the present study, we found that the PV was composed of more than one bundle (2–4 bundles; Figs. 1–6). The diameter of each bundle was 50–100 µm, and the bundles of the PV were demarcated by the boundaries of higher CT contrast (Figs. 2 and 3). In the SEM images (Fig. 6), the terminal part of the PV is considered to be a bundle composed of 8 smaller threads of ~5 µm. This finding is consistent with previous reports [1, 2, 5, 7, 9, 17] in that the PV is composed of multiple subvessels. Furthermore, the diameter of the smaller threads of the terminal PV are comparable with that of the subvessels of the PV reported by Shin et al. [9] (6.5 µm), Lee et al. [17] (10 µm), and Lim et al. [8] (10 µm). A meaningful difference from previous reports is that the proximal part of the PV was found to be composed of multiple bundles, which showed common structural features, including the diameter and number of subvessels, as well as higher CT contrast along the boundary. This pattern was also found in the PVs within the PN, where the bundles were demarcated by higher CT contrast (Figs. 2 and 3) or mesothelial cells (Figs. 5 and 6). Collectively, the results show that the PV can exist as an oligomer of primo bundles as in its proximal part and as a monomer of a single bundle as in the terminal part.

4.2. Primo subvessels and sinuses

In the cross-sectional images of the osPVS tissue scanned by micro-CT, we identified multiple circular low-density areas (30–60 a.u.; Figs. 2 and 3; marked by “2” in Fig. 3) with a tube-like structure (Fig. 2) within the PN and PV. The number of low-density areas in the PV was 10–19 in the PV of the PVS tissue shown in Fig.1. In the SEM image, at least eight subvessels were identified in the terminal PV (Fig. 6). These numbers are comparable with the number of subvessels (ductules) per unitary PV (1–20) reported by Kim [1, 2, 5]. Because no other vessel-like structure of such density/frequency is known in the PV, it is likely that the low-density areas in the CT images are indeed the primo subvessels (ductules) that have been reported previously [1, 2, 5]. However, the diameter of the low-intensity areas obtained from the micro-CT images was 2.9–3.4 µm on the long axis and 1.5–1.8 µm on the short axis, whereas the diameter of the vessel-like structures was 10~20 µm in the H&E-stained section of the PN. Considering that the reported diameter of the subvessels was 5–15 µm [1, 2, 5] and 6.5 µm [9], the diameters of the low-intensity areas are rather small. These smaller values may arise from differences in the methods used and/or shrinkage of the tissues during handling of the tissues for imaging [26, 27]. Alternatively, the round subvessel-like structures in the H&E image (Fig. 5) may represent a minor portion of the primo subvessels because they were collapsed in sample preparation, and thus, the thin flattened subvessels of small size (5–15 µm) might not be readily identifiable in the H&E images without counter-staining with specific markers. The discrepancy in the size and location of the primo subvessels may arise from the differences in sample preparation, but remains to be studied further in the future.

In micro-CT images, there were intermediate-intensity belts (yellow–green; 1-µm thickness; marked by “3” in Fig. 3) that surrounded the higher-intensity area at the boundaries of the unitary PV. The medium-intensity belts (marked by “3” in Fig. 3) tightly covered the outer boundaries (high-intensity areas, marked by “4” in Fig. 3) of the PV and inner circular areas. The location of the high-intensity areas (boundaries) overlapped with the boundaries with higher cellularity [8]. The high-intensity areas seemed to correspond to mesothelial cell layers (Figs. 4 and 6C).

Finally, the areas of lowest intensity (black) are likely to be the sinuses of the organ-surface PN, as reported previously [16, 17]. The sinuses in the PN bundle were readily identifiable, as shown in Figs. 2, 3, and 5. We did not observe well-developed sinuses in the PV bundles in the CT and H&E-stained images, but whether the sinuses in the PN bundles extend to the terminal PV is presently unknown. Taken together, among the four representative areas revealed by micro-CT image scanning, the dark areas (0 a.u.), low-intensity areas (30–60 a.u.), and high-intensity areas (130–180 a.u.) are likely to be the sinuses, subvessels, and surface layers of the PVS tissue, respectively. However, much further study is needed to understand the biological entities comprising these structures, including the intermediate-intensity areas on micro-CT images.

4.3. Primo capsule

In the CT images, the boundaries of the PVS bundles showed higher intensity (Figs. 2–4). The higher intensity boundary is likely to be associated with the surface composed of small round cells, the flat oval mesothelial cells, and the amorphous extracellular substances (Fig. 6; [15, 18]). In this study, the mesothelial cells were observed on the surface of the primo bundles in the H&E-stained section, but they were found scattered on the surface of the PVS tissue in the SEM images. In addition, the structure like the capsule reported by Vodyanoy [7] was not identified. These discrepancies might arise from the differences in processing the tissues for light and electron microscopies and remain to be studied further.

4.4. SEM images of organ-surface primo tissue

This study newly showed the whole-tissue image of a large primo tissue (length: ~4.5 cm) at lower magnification and then the detailed images of PN, PV, bundle, surface cells, and subvessels at higher magnification by SEM. This study also revealed longitudinal and circular fibrous structures on the surface of the small PV (single bundle PV; Fig. 6). As discussed earlier, the results of SEM analyses are in good agreement with the micro-CT findings shown in Figs. 1–3 and those reported in the previous reports by SEM [15, 17, 18] and light microscopy [9, 15]. For example, one can identify the primo bundles and subvessels, mesothelial cells that demarcate the primo bundle, various cells, and amorphous extracellular matrix on the surface of the primo tissue. In particular, the subvessels shown in Fig. 6D (3–7 µm) are comparable with those in Fig. 3 (~10 µm) of the study by Lee et al. [17] in size and number. It is a puzzle that both figures did not show the mesothelial cells [15] surrounding the bundle of subvessels. On the other hand, the subvessels in this study look more relaxed than those in the study by Lee et al [17], and the amorphous extracellular matrix in the two figures looks different. The latter differences are likely due to the differences in the experimental procedures in two studies. The lack of mesothelial cells in these SEM images on the primo bundle remains to be studied further.

4.5. Plasticity of primo tissue

The size and number of the PN are known to increase by more than two times in animals with anemia [19, 21] and heart failure [20]. Underlying mechanisms of such enlargement are presently not well known. In this study, the CT imaging of the enlarged PN from an anemic rat revealed that the enlargement was mostly associated with an increase in the area of intermediate intensity in micro-CT images. In considering the increase in the number of mature and immature erythrocytes in anemic PVS tissue [21], the intermediate-intensity area is likely to be due to erythrocyte proliferation. Collectively, our findings from micro-CT imaging confirmed the previous reports on anemia-induced enlargement of the PN [19–21] and further provided the possible mechanism of such plasticity.

4.6. Similarity with other network systems

The PV may exist as a multimer at the proximal part, branching out into unitary PVs, and as a monomer at the terminal part. These features of the PVS are different from those of the blood and lymphatic vascular systems. However, the structural plan of the PVS seems not to be totally new. The primo subvessels and surface layers of the PV bundles include mesothelial cells, similar to the structural plan of individual nerve axons and perineurium that covers bundles of individual nerve fibers [28]. However, the PVS is different from nerve fibers in that the capsules like the epineurium in nerve fibers are not found. In addition, the PVS is similar to the lymphatic system in that both systems have multiple nodes that are plastic in size [20, 21]. An important topic for future research would be to understand the underlying mechanism of the anemia-induced increase in the intermediate-intensity area (Fig. 4). It would also be interesting to further elucidate the differences in transmission of signals and substances between the PV and the nerve fascicle and between the nodes in the PVS and the lymphatic systems.

4.7. Limitations

The images of the PVS tissues in this study were obtained in vitro after isolation from the living body. Therefore, the orientation of the PN, PV, and smaller branches reported in this study, in particular in Fig. 6, is likely to be much different from that in vivo. The images in Fig. 1 at 90 and 180° were flattened owing to the suppression of the tissue by the Styrofoam support used for mounting the PVS tissue during micro-CT scanning. In addition, the CT images in this study were obtained without staining and with an air background based on the analyses of micro-CT images from a limited number of tissues. The resolution of micro-CT images would have been better if we adopted optimal conditions for fixation, staining of the tissue, and micro-CT scanning by taking full advantage of the micro-CT technique [23, 29, 30].

5. Conclusions

Collectively, the micro-CT analyses of the osPVS tissue revealed that a PV has one or more primo bundles of similar structure, and each primo bundle is composed of multiple primo subvessels (ductules). A PN has several enlarged primo bundles in isolation or in anastomosis. The results of this study demonstrate that the morphology of the osPVS tissue is unique among the other whole-body network systems such as the blood, lymphatic, and nervous systems. It is interesting that the structural plan of the PVS is similar to that of the lymphatic system in one aspect (i.e., nodes) and to that of the peripheral nerves in another aspect (i.e., bundles and fascicles). Our findings on the morphology of the PVS will facilitate future studies on the structure and the function of the PVS, including the biological entities contained in the intermediate-intensity areas in the CT images.