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. The first observation that LVs can have PVs inside was made by Kim in the 1960s, 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. 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. 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. 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., 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. where also LVs with a PV inside were documented. The study was done with rats. In a study by Lee and Soh, 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. reported the discovery of an LV from mouse tumor tissue with a PV inside. Shin et al. 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.. 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 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.