Coordination between intrinsic and extrinsic mechanism in thoracic lymphatics.

The lymphatic system runs in parallel with the blood vasculature, it plays a key role in maintaining tissue fluid homeostasis, as a tissue-drainage system, and it contributes to the immunosurveillance by providing a route for migrating cells. The lymphatic system is a highly branched network of thin-walled blind-ended vessels, which drain fluid, macromolecules and cells from the extracellular spaces within most organs, carrying them into larger thicker-walled collectors running deeper in the body. Fluid and solutes extravasated from vascular capillaries into the interstitial space enter blind-ended initial lymphatics, which are anchored to the interstitial matrix via anchoring filaments and possess overlapping endothelial cell-cell junctions behaving like valve structures, only permitting unidirectional lymph entry into the lymphatic vessel lumen. Valves in collecting lymphatics consist of two modified adjacent endothelial cell leaflets which meet in the vessel lumen forming a funnel inside the vessel and separating adjacent lymphangions, the functional units of the lymphatic system. Lymph is formed along a hydraulic pressure gradient developing between the interstitial tissue and the lumen of initial lymphatics. This pressure gradient depends upon both extrinsic and intrinsic pump systems. Tissue movements provide the extrinsic factor affecting lymphatic function, causing cycles of external compression/expansion of the lymphatic vessels lumen.. Lymphangions, segments of lymphatic vessel delimited by unidirectional valves and surrounded by smooth muscle cells, represent the functional units of the intrinsic pump mechanism. Their rhythmic active contraction is essential to guarantee the correct lymph flow either as the only source of pressure gradient formation or along with the extrinsic pump, where the mechanical features of the surrounding tissue are able to generate such an external pump action. During active contraction, lymphatic smooth muscle cells create an increase in intraluminal pressure and generate a local positive pressure gradient which drives lymph propulsion. The subsequent relaxation of the smooth muscle layer generates a decrease in intraluminal pressure which drives lymph from the interstitial space into the vessel itself. The aim of the present thesis was to study the interaction between the intrinsic and extrinsic mechanisms in a highly moving tissue such as the diaphragm. By in vivo fluorescence staining of diaphragmatic lymphatics we were able to identify vessels organized in loop structures and located both in the tendineous and in the peripheral muscle region. Lymphatic loops were classified into four groups (active, hybrid, passive and invariant) according to their functional behavior, forming functionally distinct regions. By whole mount immunostaining against smooth muscle actin we identified a dense smooth muscle mesh surrounding actively pumping sites, whereas in not contracting tracts smooth muscle fibers were more sparsely organized, showing a lot of large gaps around the vessel wall. Actively pumping lymphatic sites did not differ in diameter from all other classes of vessels. We found that their amplitude of contraction was independent on vessel size but strongly correlated to contraction frequency. By temporal analysis we were also able to identify trigger sites which controlled the diameter change of both other active and passive sites belonging to the same network. We then made an extensive study on the temporal correlation of activity among active, hybrid and passive sites belonging to the same network, and were able to identify trigger regions and follower regions whose behavior was dependent upon their respective trigger sites Lastly, we started an ongoing project in order to understand the extrinsic pump effect due to respiratory and cardiogenic movements on diaphragmatic lymphatic function. By locally injecting KCl into the interstitium next to invariant longitudinal and/or transverse lymphatics we tested diameter and/or length changes and then intraluminal pressure gradients due to extrinsic forces. Further analysis are required in order to define the actual contribution of intrinsic and extrinsic mechanisms in diaphragmatic lymphatics.