The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-μm microspheres as cell phantoms on slides and in 90-μm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Background and Purpose: Doxorubicin (DOX) is a risk factor for arm lymphedema in breast cancer patients. We reported that DOX opens ryanodine receptors (RYRs) to enact “calcium leak,” which disrupts the rhythmic contractions of lymph vessels (LVs) to attenuate lymph flow. Here, we evaluated whether dantrolene, a clinically available RYR1 subtype antagonist, prevents the detrimental effects of DOX on lymphatic function.Experimental Approach: Isolated rat mesenteric LVs were cannulated, pressurized (4–5 mm Hg) and equilibrated in physiological salt solution and Fura-2AM. Video microscopy recorded changes in diameter and Fura-2AM fluorescence tracked cytosolic free calcium ([Ca2+i]). High-speed in vivo microscopy assessed mesenteric lymph flow in anesthetized rats. Flow cytometry evaluated RYR1 expression in freshly isolated mesenteric lymphatic muscle cells (LMCs).Key Results: DOX (10 μmol/L) increased resting [Ca2+i] by 17.5 ± 3.7% in isolated LVs (n = 11). The rise in [Ca2+i] was prevented by dantrolene (3 μmol/L; n = 10). A single rapid infusion of DOX (10 mg/kg i.v.) reduced positive volumetric lymph flow to 29.7 ± 10.8% (n = 7) of baseline in mesenteric LVs in vivo. In contrast, flow in LVs superfused with dantrolene (10 μmol/L) only decreased to 76.3 ± 14.0% (n = 7) of baseline in response to DOX infusion. Subsequently, expression of the RYR1 subtype protein as the presumed dantrolene binding site was confirm in isolated mesenteric LMCs by flow cytometry.Conclusion and Implications: We conclude that dantrolene attenuates the acute impairment of lymph flow by DOX and suggest that its prophylactic use in patients subjected to DOX chemotherapy may lower lymphedema risk.

Pharmacological openers of adenosine triphosphate-sensitive K + (K ATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema limit their clinical usefulness. It is presumed that the arterial dilation induced by K ATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, K ATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic K ATP channel opener cromakalim (0.01-3 µmol/L) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/L) had similar effects at clinically relevant plasma concentrations. High speed in-vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/L) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time PCR and flow cytometry identified the abundant K ATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory SUR2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K + currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. Significance Statement Potassium channel openers (KCOs) are potent antihypertensive medications, but off-target effects including severe peripheral edema limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the K ATP channels in lymphatic muscle cells may be unique from their counterparts in This article has not been copyedited and formatted. The final version may differ from this version.

More than one billion people worldwide suffer from hypertension, which is a leading cause of stroke and cardiovascular disease. According to the National Heart, Lung, and Blood Institute (NHLBI) Working Group, an estimated 40% to 85% of hypertensive patients exhibit salt‐sensitivity, which increases the risk for cardiovascular events and mortality. Salt‐sensitive hypertension is known to be caused by an imbalance between sodium retention and excretion in the kidney. However, the mechanisms that enable the kidney to deliver excessive re‐absorbed salt and volume back to the systemic circulation remain obscure. Interestingly, recent studies indicate that the peripheral lymphatics act as a “salt sink” to store excessive salt and volume, thereby maintaining sodium homeostasis to regulate blood pressure. This finding led us to consider whether renal lymphatics contribute to excessive sodium retention in the kidney during the development of salt‐sensitive hypertension. To test this hypothesis, we initially evaluated lymph‐angiogenesis in the kidneys of rats with or without salt‐sensitive hypertension. Real‐time PCR, immunohistochemistry and Western blots revealed increased expression of the lymphatic‐specific marker VEGFR3 in the kidneys of Dahl salt‐sensitive (DSS) rats fed a high‐salt diet compared to either DSS rats fed regular chow or Dahl salt‐resistant (DSR) rats with or without high‐salt intake. To explore the impact of renal lymph‐angiogenesis on blood pressure, we surgically ligated the renal collecting lymph vessel (LV) in DSS rats. Ligation of the renal LV in DSS rats attenuated the increase in systolic blood pressure caused by a high‐salt diet. Notably, a growing body of evidence has implicated macrophages in lymph‐angiogenesis. Indeed, we also observed that a high‐salt diet promoted renal infiltration of macrophages, which stimulated renal lymph‐angiogenesis via upregulating VEGFR3 and its ligand VEGFC. Collectively, our data suggest that in response to a high‐salt diet, kidney‐infiltrated macrophages stimulate renal lymph‐angiogenesis, which appears to support the development of salt‐sensitive hypertension.Support or Funding InformationSupported by NIH grants T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR), and a Medical Research Endowment Award (SM) from the Univ. of Arkansas for Medical Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.