Animal models for the study of lymphatic insufficiency

Lymphedema is the term commonly employed to describe the spectrum of pathological states that arise as a consequence of functional lymphatic insufficiency. These human disease entities currently lack an effective cure. Satisfactory therapeutic strategies for both primary and secondary lymphedema will require additional insight into the complex cellular mechanisms and responses that comprise both normal lymphatic function and its regional derangement in states of pathologic dysfunction. Such insights must, initially, be derived from suitable animal models of the chronic human disease process. Historically, efforts to replicate the untreated disease of human lymphedema in animals, through surgery, irradiation, and toxicology, have been fraught with difficulty. The major impediments to the creation of satisfactory animal models have included an inability to reproduce the chronic disease in a stable, reproducible format. Recently, with the promise of potentially successful growth factor-mediated therapeutic lymphangiogenesis, and with the enhanced availability of investigative tools to assess therapeutic responses to molecular therapies, there has been a resurgence of interest in the development of viable animal models of lymphatic insufficiency. Current research has led to the development of genetic and postsurgical models of lymphedema that closely simulate the human conditions of primary and secondary lymphatic insufficiency, respectively. Such models will help to refine the assessment of various therapeutic approaches and their potential applicability to human disease interventions.     

Functional recovery of fluid drainage precedes lymphangiogenesis in acute murine foreleg lymphedema

Secondary lymphedema in humans is a common consequence of axillary lymph node dissection (ALND) to treat breast cancer. It is commonly hypothesized that lymphatic growth is required to increase fluid drainage and ameliorate lymphedema. Although there is a pronounced alteration in the balance of interstitial forces regulating fluid transport that sustains the chronic form of lymphedema, it is presently unknown whether changes occur to the balance of interstitial forces during acute lymphedema that may play a role in the recovery of fluid drainage. Here, we compared the relative importance of lymphangiogenesis of lymphatic vessels and interstitial flows for restoring fluid drainage and resolving acute lymphedema in the mouse foreleg after ALND. We found that removal of the axillary lymph nodes reduced lymph drainage in the foreleg at days 0 and 5 postsurgery, with fluid tracer spreading interstitially through subcutaneous tissues. Interstitial fluid drainage returned to normal by day 10, whereas functional regrowth of lymphatic vessels was first detected by indocyanine green fluorescence lymphography at day 15, demonstrating that the recovery of interstitial fluid drainage preceded the regrowth of lymphatic vessels. This was confirmed by the administration of VEGF receptor-3-neutralizing antibodies, which completely blocks lymphatic regrowth. It was found that the recovery of interstitial fluid drainage and the natural resolution of acute lymphedema produced by ALND were not hindered by VEGF receptor-3 neutralization, demonstrating that interstitial fluid drainage recovery and the resolution of acute lymphedema are lymphangiogenesis independent. The data highlight the central role of the interstitial environment in adapting to lymphatic injury to increase fluid drainage.     

Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.     

Multiscale modeling of lymphatic drainage from tissues using homogenization theory

Lymphatic capillary drainage of interstitial fluid under both steady-state and inflammatory conditions is important for tissue fluid balance, cancer metastasis, and immunity. Lymphatic drainage function is critically coupled to the fluid mechanical properties of the interstitium, yet this coupling is poorly understood. Here we sought to effectively model the lymphatic-interstitial fluid coupling and ask why the lymphatic capillary network often appears with roughly a hexagonal architecture. We use homogenization method, which allows tissue-scale lymph flow to be integrated with the microstructural details of the lymphatic capillaries, thus gaining insight into the functionality of lymphatic anatomy. We first describe flow in lymphatic capillaries using the Navier-Stokes equations and flow through the interstitium using Darcy's law. We then use multiscale homogenization to derive macroscale equations describing lymphatic drainage, with the mouse tail skin as a basis. We find that the limiting resistance for fluid drainage is that from the interstitium into the capillaries rather than within the capillaries. We also find that between hexagonal, square, and parallel tube configurations of lymphatic capillary networks, the hexagonal structure is the most efficient architecture for coupled interstitial and capillary fluid transport; that is, it clears the most interstitial fluid for a given network density and baseline interstitial fluid pressure. Thus, using homogenization theory, one can assess how vessel microstructure influences the macroscale fluid drainage by the lymphatics and demonstrate why the hexagonal network of dermal lymphatic capillaries is optimal for interstitial tissue fluid clearance.     

Practical aspects of indirect lymphography and lymphoscintigraphy

The objective of this presentation is to discuss practical issues related to the use of indirect lymphography and lymphoscintigraphy in the differential diagnosis of lymphedema. Indirect lymphography demonstrates the filling of initial lymphatics and of lymph collectors. Lymphoscintigraphy is utilized to generate morphological and functional imaging information about the lymphatic drainage. For the functional evaluation of lymphatic drainage, quantitative measurement of the lymph node uptake can be performed. The practical implications of these diagnostic interventions are discussed.     

Emilin1 deficiency causes structural and functional defects of lymphatic vasculature

Lymphatic-vasculature function critically depends on extracellular matrix (ECM) and on its connections with lymphatic endothelial cells (LECs). However, the composition and the architecture of ECM have not been fully taken into consideration in studying the biology and the pathology of the lymphatic system. EMILIN1, an elastic microfibril-associated protein, is highly expressed by LECs in vitro and colocalizes with lymphatic vessels in several mouse tissues. A comparative study between WT and Emilin1-/- mice highlighted the fact that Emilin1 deficiency in both CD1 and C57BL/6 backgrounds results in hyperplasia, enlargement, and frequently an irregular pattern of superficial and visceral lymphatic vessels and in a significant reduction of anchoring filaments. Emilin1-deficient mice also develop larger lymphangiomas than WT mice. Lymphatic vascular morphological alterations are accompanied by functional defects, such as mild lymphedema, a highly significant drop in lymph drainage, and enhanced lymph leakage. Our findings demonstrate that EMILIN1 is involved in the regulation of the growth and in the maintenance of the integrity of lymphatic vessels, a fundamental requirement for efficient function. The phenotype displayed by Emilin1(-/-) mice is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein and identifies EMILIN1 as a novel local regulator of lymphangiogenesis.     

Morphofunctional reorganization of the kidneys in lymphatic outflow disturbance

In 115 Wistar male rats structures and rates of tissue blood flow have been studied in the cortical and medullary renal substance histologically, polarographically (estimation of the volumetric tissue blood flow by hydrogen clearance). Systemic arterial (peritoneal aorta), venous (caudal vena cava) and lymphatic (renal lymph nodes) pressures have been measured, normal and after ligation of the thoracic duct at early (1-3 days), middle (1 month) and late (2-3 months) periods. In 1-3 days edema and dystrophy of the renal parenchyma, decrease of the blood flow rate in the cortical and its increase in the renal medullary substance, as well as a sharp elevation of pressure in the lymph nodes are observed. In 1 month of the experiment together with dystrophy and edema moderate sclerosis, decreasing blood flow rate in the cortical and medullary substance are noted. Increase of the systemic arterial and venous pressure and decreasing pressure in the lymph nodes, as well as a sharp increase of the renal nodes mass are revealed. In 2-3 months of the experiment, together with sclerosis of the renal parenchyma, elevated blood flow rate is observed in the kidneys and decreasing pressure in the lymph nodes up to its initial value takes place.     

肢体淋巴水肿磁共振淋巴造影技术方法的实验研究

目的:研究肢体水肿演变过程中不同时期的MR淋巴造影影像特征及其病理基础,探讨MR淋巴造影在肢体淋巴水肿方面的诊断价值。方法:用改良的Danese手术方法在20只新西兰大白兔后肢一侧形成淋巴水肿模型,另一侧作为对照。在每只大白兔双侧后肢足背部趾蹼处注射0.2ml欧乃影,于淋巴水肿演变过程的不同时期进行三维MR淋巴造影。结果:MR淋巴造影能准确地确定淋巴管阻塞的部位,反映淋巴管形态、功能的状况。肢体淋巴水肿的不同时期,由于其病理基础不同,产生不同的MR淋巴造影表现。结论:间质MR淋巴造影可以在解剖背景下敏感而又可靠地显示各期肢体淋巴水肿。     

Probing the effect of morphology on lymphatic valve dynamic function

The lymphatic system is vital to the circulatory and immune systems, performing a range of important functions such as transport of interstitial fluid, fatty acid, and immune cells. Lymphatic vessels are composed of contractile walls and lymphatic valves, allowing them to pump lymph against adverse pressure gradients and to prevent backflow. Despite the importance of the lymphatic system, the contribution of mechanical and geometric changes of lymphatic valves and vessels in pathologies of lymphatic dysfunction, such as lymphedema, is not well understood. We develop a fully coupled fluid–solid, three-dimensional computational model to interrogate the various parameters thought to influence valve behavior and the consequences of these changes to overall lymphatic function. A lattice Boltzmann model is used to simulate the lymph, while a lattice spring model is used to model the mechanics of lymphatic valves. Lymphatic valve functions such as enabling lymph flow and preventing backflow under varied lymphatic valve geometries and mechanical properties are investigated to provide an understanding of the function of lymphatic vessels and valves. The simulations indicate that lymphatic valve function is optimized when valves are of low aspect ratio and bending stiffness, so long as these parameters are maintained at high enough values to allow for proper valve closing. This suggests that valve stiffening could have a profound effect on overall lymphatic pumping performance. Furthermore, dynamic valve simulations showed that this model captures the delayed response of lymphatic valves to dynamic flow conditions, which is an essential feature of valve operation. Thus, our model enhances our understanding of how lymphatic pathologies, specifically those exhibiting abnormal valve morphologies such as has been suggested to occur in cases of primary lymphedema, can lead to lymphatic dysfunctions.     

A Tale of Two Models: Mouse and Zebrafish as Complementary Models for Lymphatic Studies

Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical importance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endothelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lymphedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in vertebrates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels.     

Effect of lymphangiography on lymphedema

Lymphangiography, using oily radiopaque Lipiodol Ultra Fluid has been shown to increase lymphedema in one-third of the patients with obstructive lymphedema and to cause lymphatic obliteration (demonstrated histologically). No effective element has been observed, although subclinical infection could not be ruled out. Allergy is another possible contributor, but the evidence from this series suggests that Lipiodol may act as a direct irritant when it is not rapidly cleared, as in the case of obstructive lymphedema, and proceed to obliteration of the residual lymphatics. On the basis of these findings, elective routine preoperative lymphangiography for lymphedema is considered strongly inadvisable.     

Lymphoscintigraphy: defining a clinical role

Radionuclide lymphoscintigraphy is a useful technique for differentiating lymphedema from other causes of swelling, and may sometimes be useful for delineating collateral lymphatics, the level of obstruction, and the presence of lymphoceles or abnormal collections of lymphatic vessels, if they communicate sufficiently with normal lymphatic vessels. Standardization of technique is important to provide better intrapatient and even interpatient comparison. Symmetry, numbers, and locations of lymphatic vessels, lymph nodes, abnormal collections, and dermal collaterals are helpful in the qualitative assessment of lymphoscintigraphy. In addition, region-of-interest analysis may be used to quantitate the clearance of the radiopharmaceutical from the injection site and its accumulation in draining lymph nodes. The constellation of findings may be used to assess the severity of the lymphatic obstruction, the involvement of clinically normal limbs, and to plan therapy.     

Spatio-Temporal Changes of Lymphatic Contractility and Drainage Patterns following Lymphadenectomy in Mice

Objective

To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis.

Background

Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis.

Methods

Changes in lymphatic “pumping” function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis.

Results

NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy.

Conclusions

Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage.     

Effect of interstitial edema on lung lymph flow in goats in the absence of filtration.

We tested the effect of interstitial edema on lung lymph flow when no filtration occurred. In 16 anesthetized open-thorax ventilated supine goats, we set pulmonary arterial and left atrial pressures to nearly zero and measured lymph flow for 3 h from six lungs without edema and ten with edema. Lymph flow decreased exponentially in all experiments as soon as filtration ceased. In the normal lungs the mean half time of the lymph flow decrease was 12.7 +/- 4.8 (SD) min, which was significantly shorter (P less than 0.05) than the 29.1 +/- 14.8 min half time in the edematous lungs. When ventilation was stopped, lymph flow in the edematous lungs decreased as rapidly as in the normal lungs. The total quantity of lymph after filtration ceased was 2.7 +/- 0.8 ml in normal lungs and 9.5 +/- 6.3 ml in edematous lungs, even though extravascular lung water was doubled in the latter (8.4 +/- 2.4 vs. 3.3 +/- 0.4 g/g dry lung, P less than 0.01). Thus the maximum possible clearance of the interstitial edema liquid by the lymphatics was 6.3 +/- 4.8%. When we restarted pulmonary blood flow after 1-2 h in four additional goats, lymph flow recovered within 30 min to the baseline level. These findings support the hypothesis that lung lymph flow originates mainly from alveolar wall perimicrovascular interstitial liquid and that the contribution of the lung lymphatic system to the clearance of interstitial edema (bronchovascular cuffs, interlobular septa) is small.     

The recovery of terminal lymph flow following occlusion

The effects of external pressure on the relative terminal lymphatic flow rate following occlusion of the lymph system were studied. Sulfur colloid tagged with 99mTc was injected into the hind thigh of dogs prior to compressive loading. Initially, the lymphatic clearance of the tracer was measured for approximately forty minutes with no applied external pressure. The terminal lymph vessels were then occluded for thirty minutes with the application of an applied external pressure of 75 mm Hg. Finally, the lymphatic clearance following occlusion was measured with the application of a nonocclusive pressure. External pressures of 0, 30, and 45 mm Hg were tested to determine the effects of post-occlusive pressure application on terminal lymphatic clearance. Results indicated that terminal lymphatic clearance did not resume for an applied pressure of 45 mm Hg following occlusion. The relative lymphatic clearance rate at an external pressure of 30 mm Hg following occlusion was 54% of the clearance rate for a 0 mm Hg applied pressure prior to lymph occlusion. The results for a 0 mm Hg external pressure following occlusion indicated a 23 percent clearance rate compared to the pre-occlusive state. A two compartment model was utilized to determine the lymphatic clearance rate per unit tissue volume of subcutaneous tissue from the experimental data for each pressure phase.     

An experimental model for chronic lymphedema

Although a multitude of operations exist for the treatment of lymphedema, none is highly successful. An experimental model that reliably and easily produces chronic lymphedema in an extremity would be useful to study treatments in a controlled and comparative manner and would enhance our understanding of the physiology and treatment of lymphedema. Many models that simulate clinical lymphedema have been described, but they suffer from cumbersome protocols, high laboratory costs, and an inconsistent yield of permanent lymphedema. We describe an experimental model for chronic lymphedema in the lower extremity of the rat that creates a lymphatic block in the groin induced by radiation treatment and one operation--surgical division of the superficial and deep lymphatics. All animals develop stable chronic lymphedema of the lower extremity within days of operation, with swelling that persists for at least 9 months. A mortality rate of 8 percent was associated with this technique. Methods for quantification of limb swelling are described, as is analysis of the lymphatic block by lymphoscintigraphic imaging of lymph channels and nodes. This model has the advantages of simplicity of technique, cost-effective use of rodent subjects, reproducibility of lymphedema, and quantification of results.     

The resolution of lymphedema by interstitial flow in the mouse tail skin

Lymphangiogenesis is considered a promising approach for increasing fluid drainage during secondary lymphedema. However, organization of lymphatics into functional capillaries may be dependent upon interstitial flow (IF). The present study was undertaken to determine the importance of lymphangiogenesis for lymphedema resolution. We created a lymphatic obstruction that produces lymphedema in mouse tail skin. The relatively scar-free skin regeneration that occurred across the obstruction allowed the progression of lymphangiogenesis to be observed and compared with the evolution of lymphedema. The role of vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3 signaling in lymphedema resolution was investigated by exogenous administration of VEGF-C or neutralizing antibodies against VEGFR-3. VEGF-C protein improved lymphedema at 15 days [reducing dermal thickness from 742 +/- 105 to 559 +/- 141 microm with 95% confidence intervals (CIs), P < 0.05] without increasing lymphatic capillary coverage (11.6 +/- 6.4% following VEGF-C treatment relative to 9.6 +/- 6.2% with 95% CIs, P > 0.50). Blocking VEGFR-3 signaling did not inhibit lymphedema resolution at 25 days (dermal thickness of 462 +/- 127 microm following VEGFR-3 inhibition relative to 502 +/- 87 microm with 95% CIs) or inhibit IF, although VEGFR-3 blocking prevented lymphangiogenesis (reducing lymphatic coverage to 0.2 +/- 0.7% relative to 8.7 +/- 7.3% with 95% CIs, P < 0.005). A second mouse tail lymphedema model was employed to investigate the ability of VEGF-C to increase fluid drainage across a scar. We found that neither neutralization of VEGFR-3 nor administration of VEGF-C affected the course of skin swelling over 25 days. These findings suggest that resolution of lymphedema in the mouse tail skin may be more dependent upon IF and regeneration of the extracellular matrix across the obstruction than lymphatic capillary regeneration.     

The lymphatic vasculature in disease

Blood vessels form a closed circulatory system, whereas lymphatic vessels form a one-way conduit for tissue fluid and leukocytes. In most vertebrates, the main function of lymphatic vessels is to collect excess protein-rich fluid that has extravasated from blood vessels and transport it back into the blood circulation. Lymphatic vessels have an important immune surveillance function, as they import various antigens and activated antigen-presenting cells into the lymph nodes and export immune effector cells and humoral response factors into the blood circulation. Defects in lymphatic function can lead to lymph accumulation in tissues, dampened immune responses, connective tissue and fat accumulation, and tissue swelling known as lymphedema. This review highlights the most recent developments in lymphatic biology and how the lymphatic system contributes to the pathogenesis of various diseases involving immune and inflammatory responses and its role in disseminating tumor cells.     

Lymph flow from murine footpad tumors before and after sublethal hyperthermia.

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starling's Law.