The Origin of Endothelial Cells in Novel Structures, Bonghan Ducts and Bonghan Corpuscles Determined Using Immunofluorescence

Images of a typical Bonghan corpuscle (dotted circle in A) and Bonghan duct (arrow in B). The novel thread-like structure was lifted from the surface of a rabbit's liver with forceps. The structures were semitransparent and had tensile force. They can be distinguished from other similar structures by some characteristics. For example, strings formed by fibrin are not so elastic, torn membranes do not have corpuscles, and lymph vessels are not separable from the organ surfaces. A more stringent test is to examine the distribution of nuclei by using acridine orange or DAPI.

The serial HE staining of Bonghan structures adjacent the immunofluorescence slides. (A) Bonghan duct (BHD) and its connected corpuscle (BHC). (B-E) Serial sections of the BHC. (F, G) are magnified views of B (bar = 100 μm). The threadlike BHD enters the BHC and divides into many branches to form a very complicated ball of subducts in a pool of other cells and extracellular matrices. The rod-shaped nuclei (arrows) in G indicate alignment of the endothelial cells of a subduct. Such an alignment is rarely seen because the subducts do not arrange themselves in order, but rather twirl around in a complicated manner. * = inlet of BHC.

CD146-positive endothelial cells on Bonghan ducts. (A-D) Rabbit aorta was chosen as a vascular endo thelial control. A and B are negative controls. C and its magnified view (D) show positive responses to the anti-CD146 antibody. Fluorescence signal is primarily on the internal elastic lamina (white arrow). E and F: CD146-positive responses on the Bonghan ducts. The positively stained cells formed many closed curves of various shapes and sizes as expected from the structure of the BHC, where the main ducts subdivided and branched into various sized subducts which twirled around (bar = 100 μm).

Podoplanin-positive lymphatic endothelial cells on Bonghan structures. (A, B) The lymphatic vessels on the portal area in the rabbit liver were chosen as positive controls. The anti-podoplanin-positive control reveals endothelial lines on lymph vessels (open arrow; A), while the negative control to anti-podoplanin showed no signal (B). (C-E) Podoplanin test of Bonghan corpuscles. Few anti-podoplanin-positive cells were seen (white arrows and rectangle; C). The same tissues were stained with DAPI, revealing the distribution of nuclei (D). Anti-podoplaninand DAPI-positive cells merged (E). Only five cells were co-positive (three white arrows and two cells in rectangle). Thus, there was minimal staining for podoplanin in the endothelial cells of Bonghan corpuscles (bar = 100 μm).