These data provide the first evidence that stretch- and imposed-flow-dependent regulatory mechanisms are greatly altered in aged TD. These alterations of active pumping mechanisms in TD appear to be related with age-related disturbances in NO-dependent regulatory pathways, and may reflect diminished lymphatic muscle contractility as well as altered lymphatic endothelium function.

Objective: To develop the techniques needed for the specific gene/protein targeting transfection experiments in isolated lymphatic vessels, we completed two major tasks: 1) optimize the experimental conditions to maintain the viability of isolated rat lymphatic vessels in culture for sufficiently long periods of time to permit knockdown or overexpression of selected proteins/genes and 2) develop effective transfection protocols for lymphatic muscle and endothelial cells in intact lymphatic vessels without nonspecific impairment of lymphatic contractile function due to the transfection protocol itself. Methods: Experimental protocols were developed for the maintenance of isolated lymphatic vessels under nonpressurized and pressurized conditions for 3Á12 days in culture and for adenoviral gene transfection of the lymphatic muscle and endothelial cells.Results: The data demonstrate the effectiveness of the newly developed experimental protocols for the maintenance of isolated rat mesenteric lymphatic vessels and thoracic duct in culture up to 3Á12 days without significant impairment of the parameters of their pumping and effective adenoviral/ GFP transfection of lymphatic endothelial and muscle cells in isolated rat mesenteric lymphatic vessels. Conclusions: These experimental techniques will extend the set of the modern experimental tools available to researchers investigating the physiology of lymphatic function.

The power and specificity of gene transfection has never been tested on lymphatic vessels (LV). We have optimized the organ culture technique to maintain the viability of isolated rat LV for 7‐9 days that would be sufficiently long to overexpress or knock down genes of interest. We used an adenovirus expressing GFP to test the transfection efficiency ‐ resulting in ~80‐90% GFP expression in lymphatic muscle cells with normal LV contraction patterns. We transfected several genes involved in the regulation of lymphatic contractility. After 72 hours of overexpression of SERCA2a in LV muscle cells, functional tests demonstrated diminished lymphatic tone (predicted lowered cytoplasmic Ca++) and ~50% higher lymphatic phasic amplitude (predicted enhanced systolic Ca++ release). We specifically knocked down of 80‐95% MLCK in LV ‐ both tone and phasic contractions were greatly diminished. Further refinement of the organ culture conditions allowed us to monitor LV up to 12 days under controlled pressure conditions. This experimental model allowed us to establish controlled, high‐pressure conditions (10 cm H2O) for 4‐5 days with first reversible signs of pump failure after 2‐3 days; the model can be used as an accessible and reasonable simulation of the lymphatic pump alterations during lymphedema.

All functions of lymphatic system require the lymph flow, which can not exist without the driving force generated by contractions of lymphatic vessels (LV). However the mechanisms regulating the lymphatic contractility and particularly the mechanisms of age‐related alterations in lymphatic pumping remain greatly under discovered. Using confocal imaging we observed the profound reduction of muscle cells in aged rat LV. In vivo flow measurements using fast video microscopy demonstrated greatly decreased basal lymph flow; maximal lymphocyte velocities in aged mesenteric LV are 4‐6 times lower than in the adult LV, contraction amplitude diminished by 50‐60%; while the contractions are irregular with long periods of inactivity. The profound inhibition of the contractile activity has been observed also in isolated aged rat thoracic duct (TD) and mesenteric LV. The basic flow/eNOS‐dependent regulation is abolished in aged TD, protein message is greatly depleted. At the same time in aged TD the substantial iNOS activation, confirmed by Western blotting and immunohistochemistry, occurs; its functional importance confirmed by pressure/flow tests. We concluded that depletion of the contractile reserves in LV in elderly diminishes their ability to provide the adequate transport of lymph during the periods of the increased volumetric loads. Support: NIH RO1 AG030578, HL070308, HL080526, TAMU CERH 5 P30 ES09106‐08.