Here we describe novel methods quantify pressure in draining lymph nodes and viscosity in lymphatic vessels and PLN of mice, and propose that they will be helpful towards elucidating the mechanisms during and after arthritic flare and responses to therapy using various murine models of RA15
. We hypothesize that both pressure of the lymph nodes and viscosity of lymph will increase during the pathogenesis of inflammatory arthritis. This pressure increase could be caused by the influx of macrophages and Bin cells into the draining lymph node6
, and the 5-fold increase in lymphatic pulsing10
. Additional lymphatic pressure could be caused by a change in viscosity due bone and joint catabolism in which extracellular matrix breakdown products and minerals are cleared in lymph. While lymphangiogenesis and draining lymph node expansion are designed to counteract this pressure increase and prevent lymphatic vessel rupture, there is likely a threshold pressure that exceeds the capacity of these compensatory mechanisms. We hypothesize that when this threshold pressure is achieved, it triggers shutdown of the lymphatic pulse, which results in the collapse of the draining lymph node. The resulting loss of lymphatic draining from the joint is manifested as an arthritic flare.
Despite its known importance as a biomarker of inflammation and tumor metastasis, very little has been done to measure the parameters of the murine lymphatic system because of the diminutive size of the animal compared to other species used for lymphatic investigations. Pressure has been measured in the lymphatic vessels and capillaries in humans, sheep and rabbits16–18
. The measurement of lymphatic pressure in small mammals is scarce, but an early study measured pressure of lymphatic vessels in mouse ear19
. However, there is no report of measuring lymphatic pressure in draining lymph nodes of mouse limbs where arthritis occurs. Recently, others have measured the pressure inside the human lymph node for detection of cancer cell metastasis in the lymph nodes. The study found that the intranodal pressure in sentinel lymph nodes without tumors was 9.1 ± 6.2 mmHg20
, very similar to our findings. Tumor-containing lymph nodes were found to have a pressure of 21.4 ± 15.4 mmHg; the rationale is that when more cells reside within the lymphatic vessels of a sentinel lymph node, it will increase overall pressure of the node.
Although we have no formal explanation for the difference in pressure seen between the ALN and PLN, one possible contributor is the remarkable difference in mass between the lymphatics of the lower vs. upper limbs of mice. As the volume of the lymphatics of the lower limb is apparently greater than the upper limb, we hypothesize that distribution over a greater surface area could result in lower pressure.
Others have measured the viscosity of lymph in dogs by collecting the lymph from the thoracic duct and using a viscometer. The viscosity of lymph was analyzed to determine if changes of lymph correlate with changes in diet21
. Unfortunately, parallel approaches are not applicable in smaller animals where the lymphatic vessels cannot be cannulated, and low lymph volumes preclude application of typical methods to determine viscosity. MP-FRAP overcomes these constraints by using optical techniques, opposed to physically measuring the viscosity of the fluid in question. This allows the viscosity of the fluid to be found without being collected so the viscosity of lymph can be measured throughout the course of the experiment.
The methods described in this paper overcome the size constraints of measuring murine lymph node pressure and lymph viscosity. Pressure can be measured by inserting a glass micropipette into the PLN while the viscosity of lymph can be measured by MP-FRAP. Experiments designed to test our hypotheses that both lymph node pressure and lymph viscosity will increase during the pathogenesis of inflammatory arthritis are currently underway using the methods described here.