From the five rats investigated, several parts of the PVS were identified in the abdominal cavity (at the surface of the intestine, the liver, and the abdominal wall). In general, half-transparent, milky-white, spots (PNs) were first found on the surface of the tissue and then picked by a tweezer. When the PNs were picked and moved, a vessel (a PV) was recognized in all instances as they were attached to the PN. In each rat, multiple PVS samples were taken and subsequently investigated under the microscope. The diameter of the PVs was in the range of approx. 50–150 µm.
This manuscript focuses on reporting one specific finding: red-colored parts of the PVS samples that were seen either directly without magnification when extracting the PVS parts (see the red PN in Fig. 1I, J) or under the microscope (Fig. 1A-H). One sample obtained with this characteristic consisted of four PNs and a PV of a total length of about 1 cm (Fig. 1A-E). The investigation of this sample under the microscope showed a surprising feature: a red tubular-like structure passing through the PN and all four PNs (Fig. 1A-E). The same feature was seen in another sample comprising a PN and a PV (Fig. 1F), and two samples of PNs (Fig. 1G, H). Also, two different PNs extracted exhibit red parts at their center (Fig. 1G, H). Thus, two types of red structures were found: a red thread-like structure (RTLS) and a red oval or round structure (RORS). An RTLS was observed passing thought PNs and a PV (Fig. 1A-F), and several RORSs were found to be located at the center of PNs (Fig. 1G, H).
Figure 1I and J show two instances where PVS samples were taken from the intestine surface. In both instances, a red-colored PN was first observed on the surface of the abdomen and was then pulled-out with the tweezer, revealing a PV attached to it. The sample shown in figure 1H corresponds to the PN shown in figure 1J, while the sample depicted in figure 1I is not shown as a microscopic image.
Figure 1D shows a zoomed-in part of the PV, clearly showing that the RTLS is an inner structure in the PV, possibly surrounded by an adventitia.
Since hemoglobin is the only red-colored chromophore available in the tissue of rats, the RTLS inside PVs and the RORS in PNs are most likely caused by the presence of erythrocytes. According to our view, there are three principle reasons for this. (i) The erythrocytes could be due to contamination of the PVS samples with blood coming from the surgical procedure. (ii) The PVS serves as a transport route for erythrocytes coming from a connection between the PVS and vascular blood vessels. (iii) The erythrocytes are produced inside the PVS as a form of extramedullary hematopoiesis. Possibility one can be discarded since the surgical procedures were performed carefully so to not contaminate the PVS samples with blood, and the characteristics of the red-colored parts are not in line with contamination: the RTLS is a continuous red line inside the PV, and the RORS is a part inside the PN. If contamination were the cause, erythrocytes would have also been on the outside of the extracted PVS samples. The second possibility cannot be discarded and might be true, in principle. Possibility three, however, seems to be the most likely one. The following reasons support this conclusion: (i) if extramedullary hematopoiesis takes place inside the PVS, there would be red parts inside (and not outside) the PVs and PNs, as observed. (ii) Previous studies showed that the PV contains a sinus and sub-PVs (s-PVs) that function as transport routes for a fluid (primo fluid, PF) as well as a large variety of macromolecules and cells, including stem cells. Specific cells (diameter: 3–5 µm) inside the PV and PN tested positive for expression of stem cell markers CD133, Oct3, Oct4, Nanog, SSEA, Sox2. (iii) At least four publications reported already about the presence of erythrocytes in samples of the PVS. Choi et al. microscopically observed cells with an appearance of erythrocytes in PN slices (see Fig. 1(B) in their paper). Han et al. also observed cells in the shape of erythrocytes in PN slices under the microscope (see Fig. 1(D) in their paper). Our group at SNU confirmed the presence of erythrocytes in PN slices by optical microscopy and fluorescence microscopy (hematoxylin and eosin (H&E) and hemacolor staining) (See Fig. 6 of their paper). In another paper by our group at SNU we reported also the observation of RTLS and RORS parts of PVs and PNs, respectively (see Fig. 53.1(d) in their paper) and provided evidence for the presence of erythrocytes in PVS samples from the intestine surface by H&E and hemacolor staining as well as transmission electron microscopy. We also showed that the occasions of PVS samples found with RTLS and RORS parts were higher in rats with heart failure (associated with anemia) compared to controls. We concluded that extramedullary hematopoiesis inside the PNs and PVs erythropoiesis is happening. In addition, we detected reticulocytes in the PVS samples, adding further support to the notion of extramedullary hematopoiesis (and erythropoiesis, in particular) happening in the PVS. This conclusion is supported by the finding of Kim et al. reporting the expression of the hematopoietic stem cell marker Thy 1 of cells inside the PVS.
The half-transparent, milky-white, spots (i.e., the PNs) are not Peyer’s patches (PPs). Compared to PNs, PPs are much larger in size and do not have a vessel connected the the the primo vessel. In addition, unlike the PVS tissue, the PP is based on the submucosal layer of the intestine, and so one cannot isolate the PP with its intact gross morphology like the PVS tissue.
To the best of our knowledge, a continuous red line (RTLS) traversing PNs and seeming to be inside the PV has not yet been documented, and our report is the first one providing microscopic images of this specific morphological aspect of the PVS.