Lymph Propulsion Research Articles

A novel method of measuring human lymphatic pumping using indocyanine green fluorescence lymphography

Lymph transportation through the body is partly controlled by the intrinsic pumping of lymphatic vessels. Although an understanding of this process is important for medical application, little is currently known because it is difficult to measure. Here, we introduce an easy, safe, and cost-effective technique for measuring lymphatic pumping in leg superficial lymphatic vessels. Readings obtained with this technique were compared with values obtained with dynamic lymphoscintigraphy. Differences in lymphatic pumping between healthy volunteers and patients with lymphedema were also investigated. Indocyanine green (ICG) fluorescence lymphography was performed by subcutaneously injecting 0.3 mL of ICG (0.5%) into the dorsum of the foot. Real-time fluorescence images of lymph propulsion were obtained with an infrared-light camera system with the individual supine or sitting. A custom-made transparent sphygmomanometer cuff was wrapped around the lower leg and connected to a standard mercury sphygmomanometer. The cuff was inflated to 60 mm Hg and then gradually deflated at 5-minute intervals to lower the pressure by 10-mm Hg steps until the fluorescence contrast agent exceeded the upper border of the cuff, indicating that the lymphatic contraction had overcome the cuff pressure. Lymph pumping pressure (P(pump)) was defined as the value of the cuff pressure when the contrast agent exceeded the upper border of the cuff. We measured P(pump) among healthy volunteers who maintained a supine position and compared these values with measurements obtained from lymphoscintigraphy. P(pump) values while sitting were also compared between 30 legs from healthy volunteers and 30 legs from lymphedematous patients. Among healthy, supine participants, P(pump) was 25.2 ± 16.7 mm Hg (mean ± standard deviation [SD]) when measured by ICG fluorescence lymphography. These values were significantly correlated with values taken using dynamic lymphoscintigraphy (r(2) = 0.54, p < .01), while 2 SDs of the mean were approximately 20 mm Hg, suggesting a substantial disagreement between the two methods (Bland-Altman plots). In the comparison of seated measurements, readings for healthy participants (P(pump) = 29.3 ± 16.0) were higher than those for lymphedematous participants (13.2 ± 14.9). ICG fluorescence is an accurate-as well as a safe, easy, and economical-method of measuring lymphatic pumping. Therefore, it may develop as a vital tool for diagnosing lymphatic malfunctions even when they are only in their formative stages. Studies that use this technique may increase our knowledge of the lymphatic system as a whole, allowing us to develop better treatments for lymphatic disorders.

Abstract 5238: Near-infrared fluorescence imaging of human lymphatics in healthy subjects and in cancer survivors

Abstract Near-infrared (NIR) fluorescence imaging was used to non-invasively image the (dys)function of the lymphatics in healthy control subjects and in subjects clinically diagnosed with unilateral lymphedema, a little understood disease characterized by chronic swelling and commonly observed in cancer survivors following therapeutic, complete nodal dissection. As part of an FDA approved feasibility trial and after informed consent, 24 control and 20 lymphedema (both hereditary and acquired) subjects received multiple intradermal injections of 25 µg of indocyanine green (total dose ≤ 400 µg) in bilateral arms or legs. The limbs were illuminated with NIR excitation light and the resulting fluorescent signal was imaged using custom, intensified CCD cameras. Lymphatic architecture and propulsive lymphatic flow from the injection sites to the nodal basin was observed. The apparent lymph velocities and propulsion periods were determined for the control subjects arms and legs as well as for the asymptomatic and symptomatic limbs of the lymphedema subjects. While the lymphatics of the control subjects consisted of well-defined vessels which propelled fluid from the injection sites to the regional nodal basin, diseased lymphatics exhibited distinct architectural malformations, such as (i) extravascular fluorescence in which the ICG diffused away from the injection site or leaked out of the lymphatic vessels, (ii) dense networks of lymphatic capillaries, (iii) tortuous lymphatic vessels, and (iv) lymphatic backflow and/or reflux. Statistical analyses of control subjects found subtle but significant differences in the left and right velocities in both arms (0.84 cm/s and 0.76 cm/s, p=8.1e-5) and legs (0.99 cm/s and 0.87 cm/s, p=0.032) but not in the periods of contralateral limbs. The difference in velocity in control arms (0.79 cm/s) and legs (0.94 cm/s) is small but significant (p=1.22e-7) while the difference in period is not. Significant differences in period exist between control (arms: 48.2 s; legs: 52.2 s) and both asymptomatic (arms: 39.6 s, p=2.68e-6; legs: 65.3 s, p=0.0022) and symptomatic (arms: 33.2 s, p=4.14e-5; legs: 72.1 s, p=0.00014) limbs. Significant differences were also found between the velocities in the control (0.94 cm/s) and both asymptomatic (0.83 cm/s, p=0.0084) and symptomatic (0.78 cm/s, p=0.0036) legs but not in the arms of control and lymphedema subjects. Results demonstrate that non-invasive NIR fluorescence imaging of microgram amounts of fluorophore can detect architectural and functional lymphatic abnormalities. The lack of significant differences between the velocities and periods of the asymptomatic and symptomatic limbs suggests that lymphedema may be a systemic disease regardless of onset etiology. Supported in parts by the American Cancer Society and the Longaberger Foundation (RSG-06-213-01-LR) and the National Institutes of Health (R01 HL092923 and U54 CA136404). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5238.

Abstract LB-277: Abnormal lymphatic drainage pathway and function in mice with lymph node metastases

Abstract The lymphatic system provides a major route of cancer cell dissemination from the primary lesion site, to regional draining lymph nodes (LNs), through the lymphatic to blood vasculatures, and ultimately to distant sites of metastasis. However, to date there has been no in vivo imaging technique available to demonstrate functional and structural changes of overall lymphatic system in response to tumor growth and metastasis. In order to elucidate whether there are spatial and temporal changes in lymphatic function and architecture during tumor progression, we employed a dynamic near-infrared (NIR) fluorescence imaging technique. C6/Lacz rat glioma cells were implanted intradermally into the tail at 1 to 2 cm caudal to the rectum or in the right hindlimb in balb/c nude mice. Mice with a tumor in the tail were imaged at three weeks post implantation and mice with a tumor in the hindlimb at two and three weeks after tumor inoculation. Dynamic NIR imaging with 200 ms exposure time was conducted after intradermal injection of indocyanine green (ICG) to the base of the mouse tail for a hindlimb tumor model and to the tail 2cm away from the tail tip for a tail tumor model. Our imaging data in mice with a tail tumor show no uptake of fluorophore in the lymphatic capillaries in the skin above the tumor, which is in agreement with previous reports. In addition, our results show greater staining of lymphatic capillary network to the distal tumor periphery, dilated and tortuous collecting lymphatic vessels, and reduced or loss of propulsive lymphatic flow. Moreover, altered lymphatic drainage patterns and abnormal lymphatic flow from the collecting lymphatic vessel to the lymphatic capillaries at the distal tumor margin were detected. In a hindlimb tumor model, the lymphatic drainage pattern transiently changed with progressive disease. Mice with either the inguinal (primary) or axillary (secondary) LN metastasis showed dilated and leaky fluorescent lymphatic vessels, which circumvented the primary tumor and often the inguinal LN with a tumor and drained to the brachial LN. In one mouse with the inguinal and axillary LN metastases, ICG-labeled lymph drained across the midline of the animal body and thus to the left axillary LN. Magnified fluorescent images also show diffuse dye patterns. In addition, reduced lymphatic contractile function was seen in mice with LN metastasis. In summary, we showed in an animal model that gross cancer metastasis to the draining LNs is accompanied by abnormal lymphatic drainage pathway as well as reduced functional lymph propulsion in comparison to tumor-free mice. Therefore, this emerging NIR fluorescence imaging technology can be used to non-invasively and quantitatively detect functional lymphatic changes associated with cancer, which may enable accurate nodal staging of cancer patients and provide new approaches to diagnoses and therapies against cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-277.

Lymphatic pacemaking

Lymphatic vessels display rhythmic constriction‐dilation cycle which is known as vasomotion. The rhythmic constrictions propel fluid through the lymphatic chambers which are connected by valves. Our previous study has shown that lymphatic smooth muscle pacemaking occurs through a mechanism that involves Ca2+ release from intracellular Ca2+ stores through inositol 1,4,5‐trisphosphate (IP3) receptor, and depolarization through L‐type Ca2+ channels. The aims of this study were to extend our study on single lymphatic chambers to understand lymph propulsion in chains of valve connected lymphatic chambers.Our study shows that phase locking of Ca2+ oscillations can occur across many lymphatic chambers and result in phase waves which manifest as slowly propagating waves of contractions within and across chambers. Our study also indicates that electrical coupling between chambers across valves is relatively poor. Numerical simulations indicate that chambers could partially interact through mechanical coupling between chambers.

Lymphatic pump-conduit duality: contraction of postnodal lymphatic vessels inhibits passive flow

Lymphangions, the segments of lymphatic vessels between valves, exhibit structural characteristics in common with both ventricles and arteries. Although once viewed as passive conduits like arteries, it has become well established that lymphangions can actively pump lymph against an axial pressure gradient from low-pressure tissues to the great veins of the neck. A recently reported mathematical model, based on fundamental principles, predicted that lymphangions can transition from pump to conduit behavior when outlet pressure falls below inlet pressure. In this case, the axial pressure gradient becomes the major source of energy for the propulsion of lymph, despite the presence of cyclical contraction. In fact, flow is augmented when cyclical contractions are abolished. We therefore used an in vitro preparation to confirm these findings and to test the hypothesis that lymphangion contraction inhibits flow when outlet pressure falls below inlet pressure. Bovine postnodal mesenteric lymphatic vessels harvested from an abattoir were subjected to an inlet pressure of 5.0 cmH(2)O and an outlet pressure that decreased from 6.5 to 3.5 cmH(2)O under control conditions, stimulated with U-46619 (a thromboxane analog) and relaxed with calcium-free solution. Under control conditions, lymphatic flow markedly increased as outlet pressure fell below inlet pressure. In this case, the slopes of the flow versus axial pressure gradient increased with calcium-free conditions (61%, n = 8, P = 0.016) and decreased with U-46619 stimulation (21%, n = 5, P = 0.033). Our findings indicate that the stimulation of lymphatic contractility does indeed inhibit lymphatic flow when vessels act like conduits.

Imaging of Lymph Flow in Breast Cancer Patients after Microdose Administration of a Near-Infrared Fluorophore: Feasibility Study

To prospectively demonstrate the feasibility of using indocyanine green, a near-infrared (NIR) fluorophore at the minimum dose needed for noninvasive optical imaging of lymph nodes (LNs) in breast cancer patients undergoing sentinel lymph node mapping (SLNM). Informed consent was obtained from 24 women (age range, 30-85 years) who received intradermal subcutaneous injections of 0.31-100 microg indocyanine green in the breast in this IRB-approved, HIPAA-compliant, dose escalation study to find the minimum microdose for imaging. The breast, axilla, and sternum were illuminated with NIR light and the fluorescence generated in the tissue was collected with an NIR-sensitive intensified charged-coupled device. Lymphoscintigraphy was also performed. Resected LNs were evaluated for the presence of radioactivity, blue dye accumulation, and fluorescence. The associations between the resected LNs that were fluorescent and (a) the time elapsed between NIR fluorophore administration and resection and (b) the dosage of NIR fluorophores were tested with the Spearman rank and Pearson product moment correlation tests, respectively. Lymph imaging consistently failed with indocyanine green microdosages between 0.31 and 0.77 microg. When indocyanine green dosages were 10 microg or higher, lymph drainage pathways from the injection site to LNs were imaged in eight of nine women; lymph propulsion was observed in seven of those eight. When propulsion in the breast and axilla regions was present, the mean apparent velocities ranged from 0.08 to 0.32 cm/sec, the time elapsed between "packets" of propelled fluid varied from 14 to 92 seconds. In patients who received 10 microg of indocyanine green or more, a weak negative correlation between the fluorescence status of resected LNs and the time between NIR fluorophore administration and LN resection was found. No statistical association was found between the fluorescence status of resected LNs and the dose of NIR fluorophore. NIR fluorescence imaging of lymph function and LNs is feasible in humans at microdoses that would be needed for future molecular imaging of cancer-positive LNs.

Noninvasive Quantitative Imaging of Lymph Function in Mice

Whereas functional lymph imaging in rodents is imperative for drug discovery of lymph therapeutics, noninvasive imaging of propulsive lymph function in rodents has not been reported previously. Herein, we present a noninvasive and rapid approach to measure lymphatic function in a rodent model using a near-infrared (NIR) dye to minimize background autofluorescence and maximize tissue penetration. Mice were dynamically imaged following intradermal (i.d.) injection of 2 to 10 microL of 1.3 mM of indocyanine green (IC-Green) into the tail and the limb. Our results demonstrate the ability to image the IC-Green trafficking from the lymph plexus, through lymph vessels and lymphangions, to the ischial nodes in the tail, and to the axillary nodes in the limb. Our results show that lymph flow velocity from the propelled IC-Green "packet" in the lymph vessels in the tail ranged from 1.3 to 3.9 mm/s and the fluorescence intensity peaks repeated on an average of every 51.3 +/- 17.4 seconds in five animals. While pulsatile lymph flow was detected in the deep lymph vessels, lymph propulsion was not visualized in the superficial lymphatic network in the tail. In axillary lymphatic imaging, propulsive lymph flow was also detected. The intensity profile shows that the lymph flow velocity ranged from 0.28 to 1.35 mm/s at a frequency ranging from 0.72 to 11.1 pulses per minute in five animals. Our study demonstrates the ability to noninvasively and quantitatively image propulsive lymph flow, which could provide a new method to investigate lymph function and its change in response to potential therapeutics.

Quantitative imaging of lymph function

Functional lymphatic imaging was demonstrated in the abdomen and anterior hindlimb of anesthetized, intact Yorkshire swine by using near-infrared (NIR) fluorescence imaging following intradermal administration of 100-200 microl of 32 microM indocyanine green (ICG) and 64 microM hyaluronan NIR imaging conjugate to target the lymph vascular endothelial receptor (LYVE)-1 on the lymph endothelium. NIR fluorescence imaging employed illumination of 780 nm excitation light ( approximately 2 mW/cm(2)) and collection of 830 nm fluorescence generated from the imaging agents. Our results show the ability to image the immediate trafficking of ICG from the plexus, through the vessels and lymphangions, and to the superficial mammary, subiliac, and middle iliac lymph nodes, which were located as deep as 3 cm beneath the tissue surface. "Packets" of ICG-transited lymph vessels of 2-16 cm length propelled at frequencies of 0.5-3.3 pulses/min and velocities of 0.23-0.75 cm/s. Lymph propulsion was independent of respiration rate. In the case of the hyaluronan imaging agent, lymph propulsion was absent as the dye progressed immediately through the plexus and stained the lymph vessels and nodes. Lymph imaging required 5.0 and 11.9 microg of ICG and hyaluronan conjugate, respectively. Our results suggest that microgram quantities of NIR optical imaging agents and their conjugates have a potential to image lymph function in patients suffering from lymph-related disorders.

Lymph flow modulation: the tricks of a performant machinery

The lymphatic system displays two unique and remarkable features: (a) it collects excess fluid from interstitial compartments whose pressure is mostly subatmospheric and propels the newly formed lymph against a pressure gradient back into the venous vascular bed; and (b) it adjusts its own activity to cope with the specific draining requirements of the tissue in relation to the microvascular filtration rate. These two targets are achieved through the development, within a single functional lymphatic unit, the lymphangion, of cyclic pressure waves that may either drive interstitial fluid into the lymphangion, allowing lymph formation, or squeeze the lymphangion content through the unidirectional intraluminar valves, thus sustaining centripetal lymph propulsion. Hence, two key questions in the unveiling of the secrets of the lymphatic system are how are intraluminar pressure waves generated and how can they be modulated in response to increased tissue fluid volume? Two mechanisms have been proposed (Aukland & Reed, 1993; Schmidt-Schonbein, 1990): (a) the extrinsic, or passive mechanism, which consists in the transmission of mechanical tissue forces elicited by cardiogenic or respiratory displacements or skeletal muscle contraction to the lymphatic vessel wall; and (b) the intrinsic mechanism, which consists instead in the spontaneous contraction of lymphatic smooth muscle cells triggered by phasic action potentials arising in pace-maker smooth muscles cells dispersed in the vessel wall (McHale & Roddie, 1976; McHale & Meharg, 1992). Under conditions of increased tissue fluid volume, both the contractile force and frequency may be modulated according to the tissue drainage requirements, up to attainment of maximal lymph flow at which tissue oedema eventually develops. Modulation of lymphatic tone and pump function has been attributed to various substances released by lymphatic endothelial cells in response to increased lymph flow and shear stress. According to Gashev et al. (2002 and 2004), increased shear stress in mesenteric and thoracic lymphatic ducts reduces end-systolic tone, ejection fraction and pumping frequency. Such a negative inotropic and chronotropic response was attributed to the shear stress-dependent release of nitric oxide (NO), which determines relaxation of the smooth muscle cells and flow-dependent vasodilatation. A completely different scenario was proposed by Koller et al. (1999). In small iliac lymphatic vessels, they found that higher perfusion pressure gradients determined an increased end-diastolic and end-systolic tone, leading to reduced amplitude of the diameter oscillation during the contractile phase, coupled with increased contractile frequency. This response implies a progressive increase in viscous resistances with increasing lymph flow, is abolished by removal of the lymphatic endothelium and depends upon prostaglandins and tromboxane, but not NO, release. The controversy between these two views is not trivial and concerns several aspects of the strategy adopted to modulate lymph function: (a) is the overall tone and flow resistance decreased or increased with high flows? (b) is high flow attained with stronger and energetically more expensive active pumping or with passive, low resistive vasodilation? (c) is the cellular response to increased flow fast (NO-mediated) or slow (prostanoids-mediated)? and, finally, (d) does the extrinsic mechanism play any role? A significant contribution to clarifying these intriguing questions is provided by Gasheva et al. (2006) in this issue of The Journal of Physiology. In this rigorous study, phasically active and phasically non-active segments of rat thoracic duct were challenged with increasing transmural pressures at no imposed flow, in the presence of the NO inhibitor l-NAME, or the cyclooxygenase inhibitor indo-methacine. Since local intraluminar fluid fluxes are generated in the lymphangion of the phasically active segments due to their own spontaneous contractile activity, this study allows the detection of the self-modulatory response of the lymphatic machinery to the shear stress generated by the intrinsic activity itself. Their data clearly demonstrate that, at any distending pressure, phasically active segments display, compared to non-contracting ones, a lower resting tone accompanied by a more efficient pumping amplitude during the contraction phase. This behaviour is entirely dependent upon NO release, while eucosanoids do not seem to be significantly involved. This finding implies that the lymphatic system attains the required efficiency by exploiting a combination of intrinsic and extrinsic mechanisms, thus attaining a high flow with reduced energy expenses. This new paradigm opens new questions concerning the mechanisms by which mechanotransduction of external tissue forces acting on the lymphatic wall and internal shear stress interfere with the self-regulatory intracellular pathways in order to achieve the required pumping efficiency. Hence, this elegant study by Gasheva et al. (2006), while providing some convincing answers, opens new perspectives on the function of the last almost unexplored section of the cardiovascular system.

Arnold Heller and the lymph pump

This article reviews studies on lymph propulsion in the lymph vessels by active contraction of the vessels, first described by Arnold Heller in 1869 in German language, and here translated into English. His observations were first confirmed by Beatrice Carrier (1926) and Howard Flory et al. (1927), and several groups were active up to World war II. Few publications appeared in the period 1940--1960, followed by increasing activity and development of new experimental techniques for use both in various experimental animals and in humans. Recently it has been shown that passive lymph flow may add to active propulsion. Both mechanisms depend on lymph formation, i.e. the uptake of interstitial fluid by the initial lymph vessels which is still not well understood.

Genesis and pathogenesis of lymphatic vessels.

The lymphatic system is generally regarded as supplementary to the blood vascular system, in that it transports interstitial fluid, macromolecules, and immune cells back into the blood. However, in insects, the open hemolymphatic (or lymphohematic) system ensures the circulation of immune cells and interstitial fluid through the body. The Drosophila homolog of the mammalian vascular endothelial growth factor receptor (VEGFR) gene family is expressed in hemocytes, suggesting a close relationship to the endothelium that develops later in phylogeny. Lymph hearts are typical organs for the propulsion of lymph in lower vertebrates and are still transiently present in birds. The lymphatic endothelial marker VEGFR-3 is transiently expressed in embryonic blood vessels and is crucial for their development. We therefore regard the question of whether the blood vascular system or the lymphatic system is primary or secondary as open. Future molecular comparisons should be performed without any bias based on the current prevalence of the blood vascular system over the lymphatic system. Here, we give an overview of the structure, function, and development of the lymphatics, with special emphasis on the recently discovered lymphangiogenic growth factors.

The structure of superficial lymphatics in the human thigh: precollectors.

Little is known about the morphology of precollectors, the lymphatic vessels connecting the absorbing and the collecting vessels, which are regarded as the initial drainage routes of lymph. The aim of this study was to describe the structural features of human precollectors. Samples of fat from around the saphenous veins were obtained from patients undergoing varicotomy, and serial sections were observed under light and transmission electron microscopy. Tridimensional reconstructions were also obtained by computer analysis. Precollectors were characterized by an irregular and discontinuous arrangement of smooth muscle cells in their wall. This arrangement was unrelated to the site of valves. When present, muscular elements were arranged helicoidally, as shown in tridimensional reconstructions. Under transmission electron microscopy, the endothelium of precollectors was similar to that of absorbing lymphatic vessels, irrespective of the presence of smooth muscle cells, and was thin, rich in pinocytotic vesicles, supported by a discontinous basal lamina, and connected by anchoring filaments to the surrounding connective tissue. Myoendothelial contacts were frequent. Valves were similar to those of collecting vessels, except for the presence of numerous zonulae adherentes connecting the characteristic "tip cells" of the free edge. Human thigh precollectors are characterized by the alternation of portions with a well-developed muscular coat and portions with an absorbing structure. These morphological features suggest that the precollectors contribute to fluid absorption and lymph propulsion. The frequent myoendothelial contacts suggest that smooth muscle contraction is regulated locally.

Effect of postural changes on human lymphatic capillary pressure of the skin.

1. The influence of postural changes on cutaneous lymphatic capillary pressure and venous pressure was measured at the dorsum of the foot in twelve healthy volunteers. Measurements were performed in the supine and sitting positions. 2. Lymphatic skin capillaries were visualized by fluorescence microlymphography with fluorescein isothiocyanate (FITC)-Dextran 150000. Subsequently a lymphatic capillary was punctured with a glass micropipette and pressure was measured using the servo-nulling technique. Lymphatic capillary pressure, venous pressure, heart and respiration rates were recorded simultaneously. 3. Mean lymphatic capillary pressure was significantly higher (P = 0.0096) in the sitting (9.9 +/- 3.0 mmHg) than in the supine (3.9 +/- 4.2 mmHg) position. There was no significant difference (P = 0.09) between lymphatic capillary pressure and venous pressure (6.8 +/- 3.4 mmHg) in the supine position. During sitting mean lymphatic capillary pressure was significantly lower (P = 0.0022) than mean venous pressure (53.3 +/- 4.1 mmHg). The smaller increase in lymphatic capillary pressure may be caused by the discontinuous fluid column in the lymphatic system and enhanced orthostatic contractile activity of lymphatic collectors and precollectors. Spontaneous low frequency pressure fluctuations occurred in 89% of recordings during sitting, which was significantly (P = 0.02) higher than in the supine position (54%). 4. The present results support the suggestion of enhanced intrinsic contractile activity of lymph precollectors and collectors in the dependent position. This mechanism is primarily responsible for the propulsion of lymph from the periphery to the thoracic duct during quiet sitting, when extrinsic pumping by the calf muscles is not active.

Brugia pahangi-Induced Contractility of Bovine Mesenteric Lymphatics Studied in Vitro: A Role for Filarial Factors in the Development of Lymphedema?

Products generated by filarial nematodes depress vascular reactivity by mechanisms involving endothelial cells. We hypothesized that comparable filarial-induced alterations might occur in lymph vessels. Experiments were designed to test the hypothesis that spontaneous contractions of bovine mesenteric lymphatics studied in vitro are altered by the filarial parasite Brugia pahangi. Rings of bovine mesenteric lymphatics were suspended in tissue baths and spontaneous contractions were evaluated for rate, rhythm, and amplitude. Rings that met inclusion criteria (rate > 1.8/min, regular rhythm, and an amplitude > 500 mg) were randomly exposed to B. pahangi or used as controls. Parasites were added to the bath at time zero. Changes in rate, rhythm, and magnitude of spontaneous contractions were evaluated every 10 min. Comparisons were made within control or Brugia-infected groups over time and between groups (B. pahangi versus controls). The presence of B. pahangi significantly depressed the frequency of spontaneous contractions when compared with controls. Control rings were stable over time, without changes in rate, rhythm, or amplitude. However, B. pahangi alter both the rate and rhythm of spontaneous contractions. Since spontaneous contractile activity is likely to be important in the propulsion of lymph, alterations of contractile activity could result in lymphedema. Thus, filarial factors may be responsible, in part, for altered lymphatic function seen in lymphedema. Pharmacologic intervention aimed toward influencing host-parasite metabolic interactions may, in this complicated scenario, prove useful.

Update on osteopathic medical concepts and the lymphatic system.

The osteopathic medical profession has long recognized the importance of the lymphatic system in maintaining health. A review of scientific studies shows much information on the mechanisms and importance of lymph circulation. Many osteopathic manipulative techniques designed to treat patients with tissue congestion are based on early research recognizing that lymph flow is influenced by myofascial compression. Osteopathic manipulative treatment of the diaphragm was substantiated when pressure differentials created by the thoracic diaphragm were shown to influence lymph flow. Current research demonstrates that autonomically mediated, intrinsic lymphatic contractility plays a significant role in lymph propulsion, supporting the use of osteopathic manipulative techniques directed at influencing the autonomic nervous system to improve lymphatic circulation. Although research provides an explanation of how osteopathic manipulative techniques influence the lymphatic system, experimentation to test the direct influence of manipulation on lymph circulation is needed. Clinical outcomes studies are also necessary to substantiate the clinical efficacy of osteopathic manipulative techniques.

Transport in lymphatic capillaries. I. Macroscopic measurements using residence time distribution theory.

We present a novel integrative method for characterizing transport in the lymphatic capillaries in the tail of the anesthetized mouse, which is both sensitive and reproducible for quantifying uptake and flow. Interstitially injected, fluorescently labeled macromolecules were used to visualize and quantify these processes. Residence time distribution (RTD) theory was employed to measure net flow velocity in the lymphatic network as well as to provide a relative measure of lymphatic uptake of macromolecules from the interstitium. The effects of particle size and injection pressure were determined. The uptake rate was found to be independent of particle size in the range of a 6- to 18-nm radius; beyond this size, the interstitial matrix seemed to pose a greater barrier. A comparison of 10 vs. 40 cmH2O injection pressure showed a significant influence on the relative uptake rate but not on the net velocity within the network (3.3 +/- 0.8 vs. 3.8 +/- 1.0 micron/s). This suggested the presence of a systemic driving force for baseline lymph propulsion that is independent of the local pressure gradients driving the uptake. This model can be used to examine various aspects of transport physiology of the initial lymphatics.

Disorders of lymph flow

Disturbances in blood capillary exchange of fluid, macromolecules, and cells across intact and abnormal microvessels and deranged lymphatic transport are integral, interacting components in disorders of tissue swelling. Lymphedema or low-output failure of the lymph circulation is often indolent for many years before lymphatic insufficiency (failure) and tissue swelling emerge and persist. Superimposed occult or overt infection (lymphangitis) are probably major contributors to progressive limb deformity (elephantiasis). Long-standing lymphedema is characterized by trapping in the skin and subcutaneous tissue of fluid, extravasated plasma proteins, and other macromolecules: impaired immune cell trafficking; abnormal processing of autologous and foreign antigens; heightened susceptibility to superimposed infection; local immunodysregulation; defective lymphatic (lymphangion) propulsion from an imbalance of mediators regulating vasomotion; soft-tissue overgrowth; scarring and hypertrophy; and exuberant angiogenesis occasionally culminating in vascular tumors (Fig. 8). In contrast to the blood circulation, where flow depends primarily on the propulsive force of the myocardium, lymph propulsion depends predominately on intrinsic truncal contraction, a phylogenetic vestige of amphibian lymph hearts. Whereas venous "plasma" flows rapidly (2-3 l/min) against low vascular resistance, lymph flows slowly (1-2 ml/min) against high vascular resistance. On occasion, impaired transport of intestinal lymph may be associated with reflux and accumulation and leakage of intestinal chyle in a swollen leg. Although the term "lymphedema" is usually reserved for extremity swelling, the pathogenesis of a wide variety of visceral disorders also may be traceable to defective tissue fluid and macromolecular circulation and impaired cell trafficking of lymphocytes and macrophages. Thus, lymph stasis, with impaired tissue fluid flow, underlies or complicates an indolent subclinical course with a long latent period and sporadic episodes of lymphangitis, which culminates in intense scarring. Examples are pulmonary fibrosis (e.g., pneumoconiosis), regional enteritis, retroperitoneal fibrosis, and perhaps chronic pancreatitis and cirrhosis of the liver. Transdifferentiation and ultimately transformation of endothelial and other vascular accessory cells during lymph stasis also may be pivotal to a wide range of dysplastic and neoplastic vascular disorders, including Stewart-Treves angiosarcoma, AIDS-associated Kaposi's sarcoma, and lymphangitic metastatic carcinomatosis. Lymphscintigraphy has now replaced conventional lymphography as the procedure of choice to corroborate the diagnosis of peripheral lymphedema, whereas MR imaging using paramagnetic and superparamagnetic contrast agents has the potential to yield huge dividends in furthering understanding of a variety of enigmatic edematous states, including lymphedema. Not only are better explanations and insights into swelling disorders likely to be forthcoming, but, equally important, these new, safe, noninvasive imaging techniques can and should be used to monitor the evolution and document the efficacy of commonly advocated operations and nonoperative remedies for defective lymph transport and function.

Effects of f-Met-Leu-Phe-induced inflammation on intestinal lymph flow and lymphatic pump behavior.

Previous studies in the literature indicate that intraenteric placement of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) evokes an intestinal inflammatory response characterized by an accumulation of interstitial fluid and increased lymph flow. Furthermore, it is known that movement of lymph away from the intestine is dependent on the rhythmic pumping of lymph by collecting lymphatics in the mesentery. The purpose of the present study was to determine whether the f-Met-Leu-Phe-induced increase in lymph formation is countered by an increase in lymphatic pump efficiency. Male Sprague-Dawley rats were anesthetized, and a segment of ileum with adjacent mesentery was exteriorized. The mesentery was positioned over an optical window, and a 100-microns collecting lymphatic was selected for study. The preparation was transferred to a video microscope, and the activity of the lymphatic pump was monitored under control conditions and during intraluminal infusion of 1 microM f-Met-Leu-Phe. Lymph propulsion by the lymphatic pump was calculated from the product of stroke volume and contraction frequency. In one group of animals, total lymph flow was determined by cannulating the lymphatic draining the ileal segment. Total lymph flow increased following f-Met-Leu-Phe placement in the intestine. The increased lymph flow was paralleled by a rise in lymphatic pumping. The rise in lymph propulsion by the lymphatic pump resulted exclusively from an increased stroke volume, inasmuch as contraction frequency did not change. The results of the present study suggest that activation of the lymphatic pump during acute inflammation may be important in preventing interstitial edema.

微小リンパ管におけるリンパ輸送機構の解析

微小リンパ管におけるリンパ輸送機構の解析

Relationships between lymphatic pump flow and total lymph flow in the small intestine

Response of collecting lymphatics to acute elevations in lymph formation was studied in vivo. Mesentery of 33 rats was prepared for intravital microscopic study, and indexes of lymph propulsion (contraction frequency, stroke volume, and ejection fraction) were monitored. In 14 rats, total lymph flow was measured by cannulating the main intestinal lymph trunk. After a control period, lymph flow was increased by hemodilution with isotonic saline. Right atrial pressure did not significantly increase during the hemodilution period. Total lymph flow increased to approximately 25 times control. Lymph propulsion (the product of stroke volume and contraction frequency) decreased in five lymphangions (group I). In 22 lymphangions, lymph propulsion increased one to five times control (group II). The remaining six lymphangions increased lymph propulsion by greater than five times control (group III). Group I possessed the largest end-diastolic diameter, highest baseline contraction frequency, and did not dilate as lymphatic filling increased. Group III possessed the smallest initial end-diastolic diameter and lowest baseline contraction frequency, and end-diastolic diameter doubled as lymphatic filling increased. The characteristics of group II were between those of groups I and III. Total lymph flow and lymph propulsion by group III lymphangions tended to agree when lymph flow was less than five times control. In more edematous conditions, total lymph flow exceeded lymphatic pump flow. The characteristics of group II were between those of groups I and III. Analysis of the location of various sized lymphangions within the lymphatic network indicated that the smaller group III lymphangions were located nearer the intestinal wall, and the larger group I lymphangions were nearer the outflow from the mesentery.(ABSTRACT TRUNCATED AT 250 WORDS)