The position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo

Abstract

Background: Until now, there has been no tool available to provide lymphatic researchers the ability to perform experiments in tissue explants containing lymphatic vessels under tissue position- and lymphatic lumen-controlled conditions.

Methods and results: In this article we provide technical details and description of the method of using the newly developed and implemented the position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo. In this study, we, for the first time, performed detailed comparative analysis of the contractile and pumping activity of rat mesenteric lymphatic vessels (MLVs) situated within tissue explants mounted in new tissue chambers and isolated, cannulated, and pressurized rat MLVs maintained in isolated vessel setups. We found no significant differences of the effects of both transmural pressure- and wall shear stress sensitivities of MLVs in tissue chambers and isolated MLVs.

Conclusions: We conclude that this new experimental tool, a position- and lymphatic lumen-controlled tissue chamber, allows precise investigation of lymphatic function of MLVs interacting with elements of the tissue microenvironment. This method provides an important new set of experimental tools to investigate lymphatic function.

FIG. 1.

Parts and an example of usage of the position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo. (A) Tissue chamber under microscope. 1. pipette holder with micropipette inserted into lymphatic vessel; 2. micromanipulator; 3. tissue chamber set in the middle of the custom made microscope stage top (4). (B) Parts of tissue chamber. 5. stainless steel base; 6. bottom part of tissue chamber; 7. top part of the tissue chamber. (C) and (D) Tissue chamber in its full assembly (side view and bottom view correspondingly). (E) Screen capture of the diameter tracking computer program with an example of MLV inside mesenteric tissue and diameter tracking window in analog mode.

FIG. 2.

Comparative analysis of contractile parameters of the MLVs located in tissue chambers and isolated MLVs at various levels of transmural pressure. (A) normalized outer diameter; (B) normalized contraction amplitude; (C) contraction frequency; (D) fractional pump flow. * indicates significant differences (p<0.05) between lymphatic contractile parameters within each vessel group at transmural pressure 1 cm H₂O and at other transmural pressures.

FIG. 3.

Comparative analysis of contractile parameters of the MLVs located in tissue chambers and isolated MLVs during the increases in imposed flow. (A) normalized outer diameter; (B) normalized contraction amplitude; (C) normalized contraction frequency; (D) normalized fractional pump flow. * indicates significant differences (p<0.05) between lymphatic contractile parameters within each vessel group at no imposed flow conditions, and conditions with presence of various imposed flow gradients.