Development of a Porcine Lymph Drainage Model to Study Particle Migration.

Background: Intra-articular wear particulate migration from the knee joint has been studied in various animal models as well as postmortem in patients who received total knee joint replacement. However, there still exists a need for a simple, yet analogous animal model for tracking the migration of wear debris from the knee joint, especially through the draining lymph nodes. Methods and Results: To fill this need, a proof-of-concept porcine model was developed for particle migration from the knee joint into the surrounding lymphatic system. Vitreous carbon particles were deposited both intra-articularly and extracapsularly in a bilateral manner to the hind limbs in pigs (n = 6). The regional/draining lymph nodes were qualitatively assessed weekly by a veterinarian by manual palpation to detect any enlargement or change in consistency when compared to the initial assessment before the surgical procedure. At 6 weeks, the draining lymph nodes were harvested and processed for histology. Microscopic evaluation showed carbon particle migration from the knee into 100% of the iliac lymph nodes, 50% of the inguinal lymph nodes, and 0% of the popliteal lymph nodes. Discussion: Overall, this study established a needed animal model for evaluating carbon particle migration to the draining lymph nodes from the knee joint.

Calcium and electrical dynamics in lymphatic endothelium.

KEY POINTS: Endothelial cell function in resistance arteries integrates Ca2+ signalling with hyperpolarization to promote relaxation of smooth muscle cells and increase tissue blood flow. Whether complementary signalling occurs in lymphatic endothelium is unknown. Intracellular calcium and membrane potential were evaluated in endothelial cell tubes freshly isolated from mouse collecting lymphatic vessels of the popliteal fossa. Resting membrane potential measured using intracellular microelectrodes averaged ∼-70 mV. Stimulation of lymphatic endothelium by acetylcholine or a TRPV4 channel agonist increased intracellular Ca2+ with robust depolarization. Findings from Trpv4-/- mice and with computational modelling suggest that the initial mobilization of intracellular Ca2+ leads to influx of Ca2+ and Na+ through TRPV4 channels to evoke depolarization. Lymphatic endothelial cells lack the Ca2+ -activated K+ channels present in arterial endothelium to generate endothelium-derived hyperpolarization. Absence of this signalling pathway with effective depolarization may promote rapid conduction of contraction along lymphatic muscle during lymph propulsion. ABSTRACT: Subsequent to a rise in intracellular Ca2+ ([Ca2+ ]i ), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼-70 mV. Acetylcholine (ACh) increased [Ca2+ ]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained 'plateau'). In striking contrast to the endothelium-derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+ -activated K+ (KCa ) channels as confirmed with PCR, persisted in the absence of extracellular Ca2+ , was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4-/- mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion.

Is There Volume Transmission Along Extracellular Fluid Pathways Corresponding to the Acupuncture Meridians?

Volume transmission is a new major communication signaling via extracellular fluid (interstitial fluid) pathways. It was proposed by the current authors that such pathways can explain the meridian phenomena and acupuncture effects. To investigate whether meridian-like structures exist in fish body and operate via volume transmission in extracellular fluid pathways, we injected alcian blue (AB) under anesthesia into Gephyrocharax melanocheir, which has a translucent body. The migration of AB could be seen directly and was recorded by a digital camera. The fish was then embedded and cut transversely to observe the position of tracks in three dimensions. Eight longitudinal threadlike blue tracks were recognized on the fish. The positions of these threadlike tracks were similar to meridians on the human body. Transverse sections showed that these tracks distributed to different layers of distinct subcutaneous loose connective tissues and intermuscular septa. Lymphatic vessels were sometimes associated with the extracellular blue tracks where the migration of AB occurred. Extracellular fluid pathways were found on fish through their transport of AB. These pathways operating via volume transmission appeared to be similar in positions and functions to the acupuncture meridians in Chinese medicine.

Synchronization and Random Triggering of Lymphatic Vessel Contractions.

The lymphatic system is responsible for transporting interstitial fluid back to the bloodstream, but unlike the cardiovascular system, lacks a centralized pump-the heart-to drive flow. Instead, each collecting lymphatic vessel can individually contract and dilate producing unidirectional flow enforced by intraluminal check valves. Due to the large number and spatial distribution of such pumps, high-level coordination would be unwieldy. This leads to the question of how each segment of lymphatic vessel responds to local signals that can contribute to the coordination of pumping on a network basis. Beginning with elementary fluid mechanics and known cellular behaviors, we show that two complementary oscillators emerge from i) mechanical stretch with calcium ion transport and ii) fluid shear stress induced nitric oxide production (NO). Using numerical simulation and linear stability analysis we show that the newly identified shear-NO oscillator shares similarities with the well-known Van der Pol oscillator, but has unique characteristics. Depending on the operating conditions, the shear-NO process may i) be inherently stable, ii) oscillate spontaneously in response to random disturbances or iii) synchronize with weak periodic stimuli. When the complementary shear-driven and stretch-driven oscillators interact, either may dominate, producing a rich family of behaviors similar to those observed in vivo.

Mechanobiological oscillators control lymph flow.

The ability of cells to sense and respond to physical forces has been recognized for decades, but researchers are only beginning to appreciate the fundamental importance of mechanical signals in biology. At the larger scale, there has been increased interest in the collective organization of cells and their ability to produce complex, "emergent" behaviors. Often, these complex behaviors result in tissue-level control mechanisms that manifest as biological oscillators, such as observed in fireflies, heartbeats, and circadian rhythms. In many cases, these complex, collective behaviors are controlled--at least in part--by physical forces imposed on the tissue or created by the cells. Here, we use mathematical simulations to show that two complementary mechanobiological oscillators are sufficient to control fluid transport in the lymphatic system: Ca(2+)-mediated contractions can be triggered by vessel stretch, whereas nitric oxide produced in response to the resulting fluid shear stress causes the lymphatic vessel to relax locally. Our model predicts that the Ca(2+) and NO levels alternate spatiotemporally, establishing complementary feedback loops, and that the resulting phasic contractions drive lymph flow. We show that this mechanism is self-regulating and robust over a range of fluid pressure environments, allowing the lymphatic vessels to provide pumping when needed but remain open when flow can be driven by tissue pressure or gravity. Our simulations accurately reproduce the responses to pressure challenges and signaling pathway manipulations observed experimentally, providing an integrated conceptual framework for lymphatic function.

Global abnormalities in lymphatic function following systemic therapy in patients with breast cancer.

BACKGROUND: Breast cancer-related lymphoedema (BCRL) is a result of interaction between several pathophysiological processes, and is not simply a 'stopcock' effect resulting from removal of axillary lymph nodes. The aim of this study was to test the hypothesis that there is a constitutional 'global' lymphatic dysfunction in patients who develop BCRL. METHODS: Lower-limb lymphoscintigraphy was performed in 30 women who had undergone axillary lymph node dissection at least 3 years previously, of whom 15 had BCRL and 15 did not. No patient had any clinical abnormality of the lower limb. The control group comprised 24 women with no history of cancer or lower-limb lymphoedema. (99m) Tc-Nanocoll was injected subcutaneously into the first webspace of each foot, followed by whole-body imaging. Scans were reported as abnormal if there was delay in lymph transport or rerouting through skin or deep system. Quantification was expressed as the percentage injected activity accumulating in ilioinguinal nodes. RESULTS: Mean(s.d.) ilioinguinal nodal accumulation at 150 min was significantly lower in women with BCRL than in those without (2·7(2·5) versus 5·9(4·8) per cent respectively; P = 0·006). Abnormal findings on lower-limb lymphoscintigraphy were observed in 17 of the 30 patients: ten of the 15 women who had BCRL and seven of the 15 who did not. None of the 24 control subjects had abnormal scan findings. CONCLUSION: Women with BCRL had reduced lower-limb lymph drainage, supporting the hypothesis of a predisposition to BCRL. A surprisingly high proportion of patients with breast cancer also demonstrated lymphatic dysfunction, despite clinically normal lower limbs. Possible explanations could be a systemic effect of breast cancer or its treatment, or an unidentified association between breast cancer and lymphatic dysfunction. REGISTRATION NUMBER: ISRCTN84866416 ( http://www.isrctn.com).

Primo Vessel Stressed by Lipopolysaccharide in Rabbits.

For tracking the primo vascular system, we observed the primo vessels in vivo in situ using the lipopolysaccharide (LPS) response in the lymphatic vessels of a rabbit. Injection of LPS (200 µg/kg) into the lymph nodes resulted in greatly stained primo vessels, which were swollen in some cases. We were able to obtain comparative images through alcian blue and diaminobenzidine staining, which clearly showed different morphologies of the primo vessels. The mechanism causing the response of the primo vessels to the injected LPS is still unclear; however, these results might be a first attempt at giving an explanation of the function of the primo vascular system and identifying the changes in the structure and function of the primo vascular system in response to an external stimulus such as an injection of LPS.

Reassessing cerebrospinal fluid (CSF) hydrodynamics: a literature review presenting a novel hypothesis for CSF physiology.

The traditional model of cerebrospinal fluid (CSF) hydrodynamics is being increasingly challenged in view of recent scientific evidences. The established model presumes that CSF is primarily produced in the choroid plexuses (CP), then flows from the ventricles to the subarachnoid spaces, and is mainly reabsorbed into arachnoid villi (AV). This model is seemingly based on faulty research and misinterpretations. This literature review presents numerous evidence for a new hypothesis of CSF physiology, namely, CSF is produced and reabsorbed throughout the entire CSF-Interstitial fluid (IF) functional unit. IF and CSF are mainly formed and reabsorbed across the walls of CNS blood capillaries. CP, AV and lymphatics become minor sites for CSF hydrodynamics. The lymphatics may play a more significant role in CSF absorption when CSF-IF pressure increases. The consequences of this complete reformulation of CSF hydrodynamics may influence applications in research, publications, including osteopathic manual treatments.

Extracorporeal shock wave therapy combined with vascular endothelial growth factor-C hydrogel for lymphangiogenesis.

BACKGROUND/AIMS: Lymphedema is a clinically incurable disease that occurs commonly after lymph node dissection and/or irradiation. Several studies have recently demonstrated that extracorporeal shock wave therapy (ESWT) could promote lymphangiogenesis associated with expression of vascular endothelial growth factor (VEGF)-C. This research concerned primarily the synergistic effect of ESWT combined with VEGF-C incorporated hydrogel (VEGF-C hydrogel) combination therapy for promoting lymphangiogenesis and ultimately alleviating lymphedema. METHODS: The VEGF-C hydrogel was applied to the injury site in a mouse model of lymphedema and then regularly underwent ESWT (0.05 mJ/mm(2), 500 shots) every 3 days for 4 weeks. RESULTS: Four weeks after the treatment, mice treated with VEGF-C hydrogel and ESWT showed signs of the greatest decrease in edema/collagenous deposits when compared with the other experimental group. LYVE-1-positive vessels also revealed that the VEGF-C/ESWT group had significantly induced the growth of new lymphatic vessels compared to the other groups. Western blot analysis showed that expression of VEGF-C (1.24-fold) and VEGF receptor-3 (1.41-fold) was significantly increased in the VEGF-C/ESWT group compared to the normal group. CONCLUSION: These results suggested that VEGF-C and ESWT had a synergistic effect and were very effective in alleviating the symptoms of lymphedema and promoting lymphangiogenesis.

Lymphatic pump manipulation mobilizes inflammatory mediators into lymphatic circulation.

Lymph stasis can result in edema and the accumulation of particulate matter, exudates, toxins and bacteria in tissue interstitial fluid, leading to inflammation, impaired immune cell trafficking, tissue hypoxia, tissue fibrosis and a variety of diseases. Previously, we demonstrated that osteopathic lymphatic pump techniques (LPTs) significantly increased thoracic and intestinal duct lymph flow. The purpose of this study was to determine if LPT would mobilize inflammatory mediators into the lymphatic circulation. Under anesthesia, thoracic or intestinal lymph of dogs was collected at resting (pre-LPT), during four minutes of LPT, and for 10 min following LPT (post-LPT), and the lymphatic concentrations of interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-γ, tissue necrosis factor α,  monocyte chemotactic protein-1 (MCP-1), keratinocyte chemoattractant, superoxide dismutase (SOD) and nitrotyrosine (NT) were measured. LPT significantly increased MCP-1 concentrations in thoracic duct lymph. Further, LPT increased both thoracic and intestinal duct lymph flux of cytokines and chemokines as compared with their respective pre-LPT flux. In addition, LPT increased lymphatic flux of SOD and NT. Ten minutes following cessation of LPT, thoracic and intestinal lymph flux of cytokines, chemokines, NT and SOD were similar to pre-LPT, demonstrating that their flux was transient and a response to LPT. This re-distribution of inflammatory mediators during LPT may provide scientific rationale for the clinical use of LPT to enhance immunity and treat infection.

Protein tyrosine phosphatase PTPN14 is a regulator of lymphatic function and choanal development in humans.

The lymphatic vasculature is essential for the recirculation of extracellular fluid, fat absorption, and immune function and as a route of tumor metastasis. The dissection of molecular mechanisms underlying lymphangiogenesis has been accelerated by the identification of tissue-specific lymphatic endothelial markers and the study of congenital lymphedema syndromes. We report the results of genetic analyses of a kindred inheriting a unique autosomal-recessive lymphedema-choanal atresia syndrome. These studies establish linkage of the trait to chromosome 1q32-q41 and identify a loss-of-function mutation in PTPN14, which encodes a nonreceptor tyrosine phosphatase. The causal role of PTPN14 deficiency was confirmed by the generation of a murine Ptpn14 gene trap model that manifested lymphatic hyperplasia with lymphedema. Biochemical studies revealed a potential interaction between PTPN14 and the vascular endothelial growth factor receptor 3 (VEGFR3), a receptor tyrosine kinase essential for lymphangiogenesis. These results suggest a unique and conserved role for PTPN14 in the regulation of lymphatic development in mammals and a nonconserved role in choanal development in humans.

Lymphatic drainage in the muscle and subcutis of the arm after breast cancer treatment.

Breast cancer-related lymphoedema of the arm (BCRL) results from impaired lymph drainage after axillary surgery. Little is known about lymphatic changes in the arm between surgery and oedema onset. We measured forearm muscle and subcutis lymph drainage in 36 women at 7 and 30 months after surgery by quantitative lymphoscintigraphy. None had BCRL initially but 19% had BCRL by 30 months. At 7 months muscle and subcutis drainage in both arms of BCRL-destined women exceeded that of non-BCRL women (P < 0.01). Muscle lymph drainage always exceeded subcutis drainage (P < 0.0001). Muscle lymph drainage in the ipsilateral arm was unimpaired relative to the contralateral arm. BCRL therefore developed in women with higher peripheral lymph flows. The major lymphatic load was generated by muscle; there was no pre-BCRL lymphatic impairment in the muscle of the ipsilateral arm. We propose that some women have a defined, constitutive predisposition to secondary lymphoedema. Specifically, women with higher filtration rates, and therefore higher lymph flows through the axilla that are closer to the maximum sustainable, are at greater risk of BCRL following axillary trauma, even following removal of 1-2 nodes.

Olive oil is more potent than fish oil to reduce septic pulmonary dysfunctions in rats.

BACKGROUND: Abdominal sepsis is frequently the cause of severe pulmonary dysfunction. Via the thoracic duct, the lung is the first organ exposed to gut-derived mediators released into the mesenteric lymph. AIM: The aim of this study is to investigate whether an enteral immunonutrition with long chain triglycerides prevents septic pulmonary dysfunctions. MATERIALS AND METHODS: Mesenteric lymph was obtained from lymph fistula donor rats during sepsis (lipopolysaccharides [LPS], 5 mg/kg i.p.) with or without enteral immunonutrition (1% of olive oil or 1% of fish oil). Sepsis lymph was then reinfused into the jugular vein of separate recipient rats. Thereafter, the lung tissue was analyzed for the distance of oxygen diffusion, inflammatory response, and cell apoptosis. RESULTS: Sepsis significantly increased TNFalpha release into the mesenteric lymph, whereas an enteral immunonutrition with olive oil significantly reduced the TNFalpha release into the mesenteric lymph by more than five-fold. Sepsis lymph induced a significant increase in alveolar wall thickness, inflammatory reaction, and apoptosis; whereas sepsis lymph collected during olive oil resorption prevented the thickening of the alveolar walls and induced only a mild inflammation, being more potent than fish oil to reduce septic pulmonary dysfunction. CONCLUSIONS: Mediators in the sepsis lymph induce pulmonary dysfunction. The lung may be protected by an enteral immunonutrition containing long chain triglycerides such as olive oil.

[The theory of lymphangion and current approaches to the pathogenesis, diagnosis and treatment of lymphedema of the lower extremities]. / Teoriia limfangiona i sovremennye podkhody k patogenezu, diagnostike i lecheniiu limfedemy nizhnikh konechnostei.

The paper describes the results of examination carried out by the Sankt-Peterburg school of lymphologists, pertaining to the structure, physiological properties and function of lymphangions responsible for active lymph transport. The problems of the pathogenesis, diagnosis and treatment of lymphedema of the lower extremities used in clinical practice are reviewed from the standpoint of the new theory. The data obtained as a result of the clinico-morphofunctional studies allowed to delineate the stages of lymphedema as dependent on the degree of lymphocytic structure and function integrity. Based on the aforesaid the new approaches to the diagnosis and selection of the treatment methods for lymphedema of the lower extremities have been formulated.