Overall, the lymphatic system is composed of various organs and tissues that support the immune system [1, 2] and take part in the regulation of fluid homeostasis [3, 4] and in the detoxification of the organism. The two main components of the lymphatic system comprise a system of blind-ended tubes, which are termed lymphatic vessels, and lymphatic tissue and organs, such as lymph nodes, thymus, spleen and tonsils, which are clusters of lymphoid follicles.

The lymphatic system are important for the regulation of the interstitial fluid volume, absorption of dietary fats and immune responses.

The pressure in blood capillaries induces filtration and, consequently, the plasma loses water to the interstitial fluid. Subsequently, the lymphatic vessels collect the fluid present in the extracellular space, move it through the organism and, finally, return it to the cardiovascular system, otherwise the blood pressure would drop too low to sustain homeostasis. When the fluid leaves the extracellular space and enters the lymphatic vessels, it is termed lymph. The interstitial fluid and lymph have a similar composition.

The breakdown products of fats generate molecules that are too large to pass through the endothelial cell layer of the blood capillaries. Therefore, dietary fats-derived molecules that are produced in the small intestine must enter the lacteals, which are small lymphatic vessels. After entering the lacteals, dietary fats-derived molecules are transported by the lymphatic vessels and delivered to the blood, along with the lymph.

The lymphatic system has a crucial role in the regulation of the immune responses. On one hand, lymphoid organs remove pathogens from the blood and lymph. On the other hand, lymphoid organs harbor many types of leukocytes. In addition, the lymphoid organs take part in programming the maturation of the leukocytes.

Lymph is accumulated in lymph-collecting vessels, which, in turn, merge to constitute larger vessels, termed lymph trunks. There are nine lymph trunks that drain lymph from various regions of the body: two lumbar trunks, two jugular trunks, one intestinal trunk, two bronchomediastinal trunks and two subclavian trunks (Table 1).

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Figure 1. Schematic representation of the draining system for the nine lymph trunks. There are two parallel circuits: the left side, which ultimately drains into the thoracic duct, and the right side, which drains into the junction of the right subclavian and right internal jugular veins. The intestinal trunk, along with the left and right lumbar trunks, drain into the cisterna chyli, which, in turn, is drained by the thoracic duct (left hand-side of the figure). The right hand-side of the figure illustrates the draining system for the right jugular, bronchomediastinal and subclavian trunks.

The lumbar trunks and intestinal trunk drain into a large vessel that is named cisterna chyli (Figure 1). Subsequently, the cisterna chyli and the remaining lymph trunks drain into one of the lymph ducts. Specifically, the cisterna chyli and the trunks of the left side of the body drain into the thoracic duct (Figure 1), which drains the entire lower part of the body and the left side of the upper body (Figure 2). The jugular, bronchomediastinal and subclavian trunks drain into small right lymphatic ducts (Figure 1), which then drain into the junction of the right subclavian and right internal jugular veins, in order to drain the upper right side of the body (Figure 2). Detailed discussions for lymphatic system in specific organs such liver [5] can be found elsewhere.

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Figure 2. This figure shows the two draining circuits of the human body: (1) the upper right-side region; (2) the remaining part, which comprise the upper left-side and the entire lower part of the body. The double arrowheads indicate the anatomical directions utilized in the figure.

There is no pump to propel the lymph within the lymphatic vessels, therefore, the lymphatic system results in a low-pressure circuit. In most cases, the lymph is moved against the gravity force, so valves must be present in the lymphatic vessels to prevent lymph backflow. In analogy with veins, lymphatic vessels are entrenched between muscles. The contraction of muscles gently presses the lymph in the direction of the heart. In most cases, the lymph is moved against the gravity. The contraction of the smooth muscles also takes part in maintaining the lymph flow. Smooth muscles are present in the walls of lymph-collecting vessels.

Lymphatic capillaries are tiny structures that assume a web-like shape, in order to encircle the blood capillary beds. In contrast to the blood capillaries, the lymphatic capillaries are blind ended. Therefore, the lymphatic vasculature is a one-way system, which has the function to move the lymph away from the tissues.

The walls of the lymphatic capillaries contain cells that have the ability to flap open and closed, as they are not tightly joined to each other. The fluids that are lost by the blood capillaries build up in the interstitial space and result in an increased fluid pressure, which pushes lymphatic endothelial cells apart. This allows large quantities of fluids to enter the lymphatic capillaries. If the interstitial fluid pressure should decrease, the lymphatic endothelial cells flap shut. The lymphatic vessels must be sufficiently leaky to allow the access of macrophages and of other types of cells of the immune system. Pathogens such as bacteria and malignant cells that might be present in the interstitial fluid also have the ability to enter the lymphatic vessels. Then, the lymphatic vessels convey the pathogens toward the lymph nodes, which are clusters of lymphoid organs that function as filters to trap pathogens, so they cannot affect other parts of the body.

The type of tissue of the lymphatic system is predominantly based on loose connective tissue, termed reticular tissue, which is made of thin reticular fibers and various kinds of specialized cells. The reticular tissues form a sort of a net that has the function to trap pathogenic agents. The lymphatic organs contain leukocytes, such as B and T lymphocytes, macrophages, dendritic cells and reticular cells. The latter are abundantly present in lymph nodes and spleen ad produce reticular fibers, which produce thin collagen proteins constitute reticular fibers. The pathogens that are trapped in the lymphatic tissues are eliminated by the leukocytes and dendritic cells. Lymph nodes have the ability to trap 90% of pathogenic agents that are present in the lymph. This prevents the transport of pathogens into the bloodstream and, consequently, the spreading to other organs and tissues. Once the pathogens have been removed, the cleaned lymph drains out through the efferent lymphatic vessels that are present on the opposite side of the lymph node, which is termed hilum.

The spleen is the largest lymphoid organ and is situated in the left upper quadrant of the abdominopelvic cavity. The color of the spleen is purplish-brown and its internal structure consists of a dense network of reticular fibers. There are two different histological regions within the reticular network: the red pulp, which harbors macrophages that eliminate old erythrocytes, and the white pulp that filters the pathogenic agents from the blood. Leukocytes and dendritic cells are present in the white pulp.

The thymus is a small encapsulated organ and is situated in the superior mediastinum. The thymus does not trap pathogenic agents. Its function consists of producing hormones that are required for the maturation of functional T lymphocytes. The thymus has two lobes. Each lobe comprises two regions: the outer cortex and the inner medulla. Most of the T lymphocytes are contained in the cortex. The medulla has much less T lymphocytes. The function of the medulla consists of destroying certain subpopulations of T lymphocytes that might have the potential to attack cells and/or tissues of the host.

In the process of embryogenesis, the lymphatic vasculature derives from the cardinal vein and then is formed independently from the circulatory system of the blood [6, 7]. The homeobox gene Prox1 has been identified as a main regulator of lymphangiogenesis, as demonstrated in experiments on mouse embryos [7]. Prox1 is expressed in a subpopulation of endothelial cells, which bud from the veins to constitute the mammalian lymphatic system. These studies were carried out by monitoring the lymphatic markers VEGFR-3, LYVE-1, chemokine (C–C motif) ligand (CCL)21 and SLC. In the absence of Prox1, the mouse embryos failed to generate the lymphatic vasculature. After the initial specification and budding from the cardinal vein, the VEGFR-3 becomes a key player in the regulation of lymphangiogenesis, in which new lymphatic vessels continue to sprout from previously generated lymphatic components.

The initial sprouting of lymphatic vessels from the cardinal vein is then followed by the maturation of lymphatic capillaries, which is regulated by the Notch signaling axis [12]. The process of maturation of the collecting lymphatic vessels is regulated by the angiopoietin 2 ANG2 and involves the formation of valves and the inclusion of smooth muscle cells [13, 14]. In fact, ANG2-deficient mice failed to develop a normal lymphatic vascular system [13]. The lymphatic capillary development is modulated by the forkhead box protein FOXC2, which coordinates inclusion of the smooth muscle cells, along with the formation of the basement membrane [9]. FOXC2 and Prox-1 work in combination for the monitoring of the lymph flow, to promote the expression of the gap junction protein connexin 37 (Cx37) and to stimulate the signaling system of the calcineurin/nuclear factor of activated T-cells (NFAT), which is required for the formation of the lymphatic valves [10].

Other factors that are involved in the regulation of lymphangiogenesis include bone morphogenetic protein 9 (BMP9), activin receptor-like kinase 1 (ALK1), sphingosine-1-phosphate (S1P)/S1P receptor (S1PR)1 and fibroblast growth factor (FGF)2/FGF receptor (FGFR)1 [8]. Lymphangiogenesis is also affected by pro-inflammatory cytokines [15]. Type 1 cytokines support lymphangiogenesis, whereas type 2 and anti-inflammatory cytokines exhibit an inhibitory effect on lymphangiogenesis [15]. Type 1 cytokines comprise IL-1, IL12, IL-18 and TNF-α. Type 2 and anti-inflammatory cytokines include IL-4, IL-5, IL13 and IL-10. Lastly, low doses of the pro-inflammatory lipid molecule leukotriene B4 (LTB4) support lymphatic regeneration, whereas high pathological concentrations of LTB4 turn down lymphangiogenesis [16].

In summary, the regulation of the process of lymphangiogenesis depends on the interstitial fluid flow and on the expression of cytokines and growth factors.

Lymphatic dysfunctions have critical implications for the onset of a variety of disorders, such as obesity [17, 18], type 2 diabetes [19, 20], atherosclerosis [21], myocardial infarction [18, 21] and immune deregulations [22].

Lymph flow is moved within lymphatic vessels in opposition to the hydrostatic pressure gradient in most regions of the organism. Lymph flow regulation requires two types of forces: extrinsic, or passive, and intrinsic, or active forces. The so-called intrinsic, or active forces, propel lymph flow through the combination of the lymphatic muscle cells contractions with the function of intraluminal valves, which, in turn, result in a synchronized contraction waves within the lymphangion. In general, the pumping of the lymph flow consists of an initial preload, afterload and synchronous contraction rate, which are regulated by the nervous system [23, 24]. The humoral modulators that regulate the lymphatic smooth-muscle contractions comprise serotonin, epinephrine and prostaglandin E1 (PGE1) [25].

The propulsion of the lymph flow is also influenced by the actions that surrounding tissues apply on the lymphatic vessel walls and lumen. In this case, the lymph flow propulsion is influenced by so-called extrinsic, or passive forces [23, 24].

A number of protocols can be utilized for the detection and measurement of the lymph flow.

Peripheral lymphoscintigraphy is a nuclear medicine technique, which can be used to visualize the peripheral lymphatics that are under the control of the autonomic nervous system [24]. This technique can also be utilized for the imaging of the blood vessels that supply a particular anatomical region. A study on patients required the subcutaneous injection of 20 MBq (0.54 mCi) Tc-99m antimony sulfur colloid (0.1 ml). The imaging was carried out with an Elscint SPWB gamma camera, along with a dedicated computer (Elscint, Haifa, Israel) for 5 minutes per frame, using a 256 x 256 matrix size [24].

Lymphangiography is older than lymphoscintigraphy and it involves the cannulation of the lymphatic vessels, in order to inject some milliliters of contrast agents, such as CT, for X-ray computed tomography, or MR, for magnetic resonance imaging [26, 27]. The CT contrast agent requires the subcutaneous injection of 1 ml of methylene bleu and 2% lidocainemixed liquor, in a 1:1 volume ratio [28], which is followed by the injection of 1% lidocaine [28]. Following the administration of the contrast agent, CT lymphangiography (CTL) can be carried out with an appropriate scanner, such as SOMATOM Sensation (Siemens Medical Healthcare, Forchheim, Germany), or Brilliance iCT (Philips Medical Healthcare, Best, The Netherlands) [28]. The MR contrast agent consists of an intracutaneous injection of 4 ml of a gandolinium-based contrast agent (gadobenate dimeglumine, MultiHance) and 1 ml of 1% lidocaine [29]. MR imaging (MRI) is carried out either with 1.5 T, or 3.0 T scanner platforms [29]. The interpretation of the examination is based on 3D image postprocessing software for the interactive MIP and MPR creation (GE Advanced Windows Workstation) [29]. However, the cannulation of the lymphatic vessels can be problematic and requires specialized technical skills, both for CTL and MRI procedures.

Lymphatic imaging can also be achieved with near-infrared fluorescence (NIR), which provides faster lymphatic function imaging than lymphangiography and/or lymphoscintigraphy, because of the enhanced sensitivity that is associated with higher photon count rates from NIR organic fluorophores than typical radiotracers [26]. An organic fluorophore is based on indocyanine green (ICG) for the imaging of the human lymphatic system. ICG is a tricarbocyanine dye, which has been utilized for hepatic and ophthalmology applications. Usually, the ICG total dose in patients is less than 25 mg. ICG solutions absorb between 760 and 785 nm and the resulting fluorescence can be imaged between 820 and 840 nm [26].

Manual lymphatic drainage (MLD) consists of a manual massage, which may improve the function of the lymphatic system, by modulating the interstitial pressures. The process only requires a light pressure that can be applied with various movements of the hands [30]. MLD may prevent the formation of edema that may result from a trauma or injury [30]. Following the development of an edema, the lymphatic system becomes a main player in taking the interstitial fluid in excess, which is then returned into the circulatory system. For instance, edemas may derive from orthopedic injuries [30], cancer treatment [31], cervical cancer [32], systemic sclerosis [33], bariatric surgery for the treatment of morbid obesity [34], burn trauma [35] and venous disease [36]. Of course, these are only some examples. There is a wide spectrum of conditions that may induce edemas.

Ultrasound therapy can also be applied for the improvement of the lymphatic system functions [37]. A clinical study showed that ultrasound therapy and MLD achieved the same results in treating women with leg swelling that was caused by wearing high-heeled shoes [37]. The ultrasonic treatment was conducted with a constant wave of 1 MHz, 1.0 W.cm, which was administered for 7.5 minutes per leg [37, 38].

Temperature can modulate the contraction frequency that propels lymph flow within the lymphatic system [39]. The effect of temperature has a direct influence on the intrinsic active spontaneous contractions of the lymphatic vessel muscle. Studies in the rat model showed a tissue-dependent differential response to the effect of temperature variation, as indicated by the analysis of excised tissues from the diaphragm and the hind paw [39]. The temperature values tested in the study were 24°C, 33°C and 40°C. A sigmoidal trend was observed between the lymphatic contraction frequency (fc) and the temperature in both vessel types, which was centered in the average temperature of the corresponding tissue that surrounds the lymphatic vessel. The diaphragmatic lymphatics peak was observed at 36.7°C, whereas the peak of the hind paw vessels was at 32.1°C [39]. Studies are underway to determine the effects of chronic variations of the temperature in lymphatic vessels of various parts of the body [39].