Immunofluorescence staining of the primo-vessel in a lung cancer xenograft.

<p>Primo-vessel cross sections were treated with anti-LYVE1 polyclonal antibody (green) or iso-type control polyclonal antibody (C, D) and observed with confocal microscopy. Lymphatic vessel cross sections were stained with the same antibodies and observed as positive controls (A, B). Fluorescence images of DAPI (blue) exhibiting nuclei and DIC images were also taken. Lymphatic endothelial cells with strong positive staining clearly located at the inner boundary of lymphatic vessel, whereas no LYVE-1 positive cells were seen in the primo-vessel. A part of the same sample which was treated with only DAPI without sectioning and mounted longitudinally revealed aligned rod-shape nuclei surrounding lumens assumed to be endothelial cells of primo-vessel (E).</p

Histological examination of the primo-vascular system in lung cancer xenografts.

<p>(A) Shown are a representative hematoxylin and eosin (H&E)-stained primo-node cross-section (top) and a Masson's trichrome (MT)-stained adjacent-section (bottom). Right figures are enlarged images of left figures. (B) Depicted are primo-vessel cross-sections which were stained with H&E (left) and MT (right). (C) H&E staining of an oblique section containing both a primo-node (a double arrow) and a primo-vessel (a dotted arrow). An image of the primo-node was magnified in the right figure. There are multi lumens (arrows) with floating cells (arrowheads) in all samples which are distinct from a single lumen structure of lymph or blood vessels. Furthermore, MT staining revealed primo-vascular system consists of loose collagen fibers (blue color) surrounding lumens.</p

Intraoperative visualization of the primo-vascular system attached to tumor nodules in i.p. lung cancer mouse models.

<p>Primo-vessels (arrows) stained with trypan blue mainly located around tumor nodules (arrowheads) which were visible after 4 weeks of i.p. injection with NCI-H460 cells. Especially some primo-vessels showed distribution patterns either going inside or surrounding tumor nodules. SI; small intestine, AW; abdominal wall.</p

Intraoperative visualization of the primo-vascular system in abdominal cavity of s.c. lung cancer mouse models.

<p><i>In situ</i> trypan blue spreading revealed semitransparent primo-vessels (arrows) which are distributed on the surfaces of various organs due to higher infiltration of the dye into loose extracellular structure of primo-vessels compare to other tissues. Straight thin primo-vessels were often visible, but sometimes many forked branches with a junction (an asterisk) were also found. Note that primo-vessel is liftable by forceps in contrast to lymphatic vessels (B). LI; large intestine, SI; small intestine, O; oviduct, L; liver, K; kidney, AW; abdominal wall.</p

Visualization 1: Monitoring the primo vascular system in lymphatic vessels by using window chambers

2 hours after AB injection Originally published in Biomedical Optics Express on 01 April 2016 (boe-7-4-1251

Visualization 2: Monitoring the primo vascular system in lymphatic vessels by using window chambers

20 hours after AB injection Originally published in Biomedical Optics Express on 01 April 2016 (boe-7-4-1251

Phase contrast microscope images of the PVS.

<p>A: The fluorescent image of the FNPs that were injected at a PN located about the CV4 and flowed in a PVS buried in the adipose tissue of the AWFB. It flowed up to the CV 14 and reemerged to the abdominal cavity toward the liver surface. The flow line was barely visible under the stereo fluorescence microscope. B & C: Phase contrast microscope images of the bright mode (B) and fluorescent mode (C) of the boxed region in (A). The PV (dashed arrow) running parallel to and above the blood vessel (two arrows) is hardly visible in (B) but clearly observable with fluorescence of FNPs in the panel (C). This primo vessel is the first observation of the so called extra vascular PVS. It runs closely along the blood vessel. The AWFB is clearly seen in (B) and its boundary is depicted with two curves in (C). The boundary of the abdominal wall fat band is indicated with two yellow curves. 40x. D & E: The PN is not noticeable without the fluorescence in (D), but manifestly appears with fluorescent view in the panel (E). The size of the PN was 250 μm. The fluorescent nanoparticles were highly concentrated in the PN. 40x</p

Mast cells in the PN.

<p><b>A:</b> The fluorescence of FNPs indicates the location of a PN (arrow). <b>B</b>: The toluidine blue staining cannot distinguish the parietal peritoneum and the PN. It showed the presence of mast cells (broken arrows) in the PN. The PN was torn off from the peritoneum during the sectioning process. <b>C</b>: An intact PN stained with toluidine blue was obtained. The overview of an AWFB-section shows a PN which are well kept just inside the parietal peritoneum. A lymph node (LN), blood vessels (BV), adipose tissues and parietal peritoneum (PP) are also seen showing their relative locations. It is a rare and fortunate case that a lymph node and a PN located nearby was found as presented in this figure. <b>D</b>: A magnified view shows that the cells in the lymph node and the PN look different. In fact, there were no mast cells in the lymph node and but many in the PN. <b>E</b>: A further magnified image depicts clearly the distribution of mast cells (*). The toluidine blue staining can be used for identifying the PN by revealing the abundance of mast cells even though it could not distinguish the collagens of the PN and surrounding connective tissues.</p

Histological analysis of the PV, peritoneum and fascia.

<p><b>A:</b> The position of the tissue block for the cross section. <b>B:</b> The spot of fluorescence (arrow) due to FNPs is the position of the PV. Its size is 10 μm and 150 μm away to the left from the large blood vessel. <b>C:</b> The H&E staining barely revealed the spot of the PV (arrow) just inside the parietal peritoneum. This figure showed that the PV and the surrounding connective tissue of the parietal peritoneum are not distinguishable with H&E. The deep fascia and the parietal peritoneum are barely distinguishable. Muscle (M) is clearly distinguished by color. B &C are the same sections. <b>D</b>: Another section showed the fluorescence spot of the PV (arrow). <b>E</b>: The Mason’s trichrome staining cannot distinguish the PV (arrow) and the parietal peritoneum. It can clearly distinguish the parietal peritoneum and the deep fascia. Muscle (M) is also well distinguished. D &E are the same sections.</p

The anatomical position of the novel flowing duct in the abdominal wall fat band.

<p>A: Schematic illustration showing the location of linea alba and the conception vessel CV in the abdominal skin side. The broken line is the surgery cutting line of the abdominal wall. The line is in the right hand side from the linea alba in order to avoid cutting the PVS in the AWFB. The FNPs that were injected to a PN entered the AWFB and appeared at the terminal point to be continued to the PVS on the liver surface. B: The blood vessels in the AWFB inside the parietal peritoneum of the abdominal wall. The locations CV 8 to 14 are mere markings for positional references and not real CV-acupoints. Note that the CV8 corresponds to the umbilicus and the parietal peritoneum continues down to the ligament wrapping the bladder. C: A PV (arrow) emerged from the AWFB (double arrows) was raised tautly with a forceps.</p