Moriondo A, Solari E, Marcozzi C, Negrini D. Lymph flow pattern in pleural diaphragmatic lymphatics during intrinsic and extrinsic isotonic contraction. Am J Physiol Heart Circ Physiol 310: H60 -H70, 2016. First published October 29, 2015 doi:10.1152/ajpheart.00640.2015.-Peripheral rat diaphragmatic lymphatic vessels, endowed with intrinsic spontaneous contractility, were in vivo filled with fluorescent dextrans and microspheres and subsequently studied ex vivo in excised diaphragmatic samples. Changes in diameter and lymph velocity were detected, in a vessel segment, during spontaneous lymphatic smooth muscle contraction and upon activation, through electrical whole-field stimulation, of diaphragmatic skeletal muscle fibers. During intrinsic contraction lymph flowed both forward and backward, with a net forward propulsion of 14.1 Ϯ 2.9 m at an average net forward speed of 18.0 Ϯ 3.6 m/s. Each skeletal muscle contraction sustained a net forward-lymph displacement of 441.9 Ϯ 159.2 m at an average velocity of 339.9 Ϯ 122.7 m/s, values significantly higher than those documented during spontaneous contraction. The flow velocity profile was parabolic during both spontaneous and skeletal muscle contraction, and the shear stress calculated at the vessel wall at the highest instantaneous velocity never exceeded 0.25 dyne/cm 2 . Therefore, we propose that the synchronous contraction of diaphragmatic skeletal muscle fibers recruited at every inspiratory act dramatically enhances diaphragmatic lymph propulsion, whereas the spontaneous lymphatic contractility might, at least in the diaphragm, be essential in organizing the pattern of flow redistribution within the diaphragmatic lymphatic circuit. Moreover, the very low shear stress values observed in diaphragmatic lymphatics suggest that, in contrast with other contractile lymphatic networks, a likely interplay between intrinsic and extrinsic mechanisms be based on a mechanical and/or electrical connection rather than on nitric oxide release. lymph propulsion; tissue stress; spontaneous lymphatic contractility NEW AND NOTEWORTHYIn diaphragmatic lymphatics, flow velocity and lymph flow were more than two order of magnitude greater during contraction of diaphragmatic skeletal muscle than during spontaneous contraction of lymphatic smooth muscles, suggesting a marginal role of the latter in setting lymph flow in rhythmically moving, thoracic tissues.THE PLEURAL DIAPHRAGMATIC lymphatic system is composed of linear lymphatic vessels preferentially located in the tendineous and the medial muscular portion of the diaphragm, and of a more complex net of loop-like structures interconnected by short linear tracts and preferentially located at the most peripheral diaphragmatic rim (18). Given the strategic role played by pleural lymphatics in setting the correct pleural fluid volume and subatmospheric pressure required to maintain the normal lung chest wall coupling (29), much effort has been spent in the study of the inner regulatory mechanisms of lymph drainage and propulsion in th...
The spontaneous contractility of FITC-dextran-filled lymphatics at the periphery of the pleural diaphragm was documented for the first time "in vivo" in anesthetized Wistar rats. We found that lymphatic segments could be divided into four phenotypes: 1) active, displaying rhythmic spontaneous contractions (51.8% of 197 analyzed sites); 2) stretch-activated, whose contraction was triggered by passive distension of the vessel lumen (4.1%); 3) passive, which displayed a completely passive distension (4.5%); and 4) inert, whose diameter never changed over time (39.6%). Smooth muscle actin was detected by immunofluorescence and confocal microscopy in the vessel walls of active but also of inert sites, albeit with a very different structure within the vessel wall. Indeed, while in active segments, actin was arranged in a dense mesh completely surrounding the lumen, in inert segments actin decorated the vessels wall in sparse longitudinal strips. When located nearby along the same lymphatic loop, active, stretch-activated, and passive sites were always recruited in temporal sequence starting from the active contraction. The time delay was ϳ0.35 s between active and stretch-activated and 0.54 s between stretch-activated and passive segments, promoting a uniform lymph flux of ϳ150/200 pl/min. We conclude that, unlike more central diaphragmatic lymphatic vessels, loops located at the extreme diaphragmatic periphery do require an intrinsic pumping mechanism to propel lymph centripetally, and that such an active lymph propulsion is attained by means of a complex interplay among sites whose properties differ but are indeed able to organize lymph flux in an ordered fashion. diaphragmatic lymphatic network; initial lymphatics; intrinsic lymphatic mechanism THE DIAPHRAGMATIC LYMPHATIC network is a complex vascular system arranged both over the pleural and peritoneal sides of the diaphragm (3, 18). Both linear lymphatics and vessels are arranged in complex loops that run parallel to the diaphragmatic surface, forming a superficial submesothelial network. Transverse ducts depart from these vessels and run perpendicular to the diaphragmatic surface through the skeletal muscle fibers to reach larger collecting lymphatics, located in the center of the diaphragmatic thickness and supposed to propel the lymph away from the diaphragm (7). Throughout the body tissues, lymph formation and propulsion have been found to rely on two different mechanisms: one intrinsic, due to the rhythmic spontaneous contraction of the smooth muscle cells of the lymphatic vessel wall (12), and the other extrinsic, associated with tissue displacements and depending on the mechanical stresses arising in the tissue surrounding the lymphatics (15). Diaphragmatic lymphatic function has been extensively studied by means of fluorescence in vivo imaging and micropuncture technique in loops and linear vessels that are located in the muscular region just below the mesothelial layer. In this superficial network of the diaphragm, both cardiac and respiratory activities...
Lymphatic vessels drain and propel lymph by exploiting external forces that surrounding tissues exert upon vessel walls (extrinsic mechanism) and by using active, rhythmic contractions of lymphatic muscle cells embedded in the vessel wall of collecting lymphatics (intrinsic mechanism). The latter mechanism is the major source of the hydraulic pressure gradient where scant extrinsic forces are generated in the microenvironment surrounding lymphatic vessels. It is mainly involved in generating pressure gradients between the interstitial spaces and the vessel lumen and between adjacent lymphatic vessels segments. Intrinsic pumping can very rapidly adapt to ambient physical stimuli such as hydraulic pressure, lymph flow-derived shear stress, fluid osmolarity, and temperature. This adaptation induces a variable lymph flow, which can precisely follow the local tissue state in terms of fluid and solutes removal. Several cellular systems are known to be sensitive to osmolarity, temperature, stretch, and shear stress, and some of them have been found either in lymphatic endothelial cells or lymphatic muscle. In this review, we will focus on how known physical stimuli affect intrinsic contractility and thus lymph flow and describe the most likely cellular mechanisms that mediate this phenomenon.
Diaphragmatic lymphatic function is mainly sustained by pressure changes in the tissue and serosal cavities during cardiorespiratory cycles. The most peripheral diaphragmatic lymphatics are equipped with muscle cells (LMCs), which exhibit spontaneous contraction, whose molecular machinery is still undetermined. Hypothesizing that spontaneous contraction might involve hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in lymphatic LMCs, diaphragmatic specimens, including spontaneously contracting lymphatics, were excised from 33 anesthetized rats, moved to a perfusion chamber containing HEPES-Tyrode's solution, and treated with HCN channels inhibitors cesium chloride (CsCl), ivabradine, and ZD-7288. Compared with control, exposure to 10 mM CsCl reduced (-65%, n = 13, P < 0.01) the contraction frequency (F) and increased end-diastolic diameter (D, +7.3%, P < 0.01) without changes in end-systolic diameter (D). Ivabradine (300 μM) abolished contraction and increased D (-14%, n = 10, P < 0.01) or caused an incomplete inhibition of F (n = 3, P < 0.01), leaving D and D unaltered. ZD-7288 (200 μM) completely (n = 12, P < 0.01) abolished F, while D decreased to 90.9 ± 2.7% of control. HCN gene expression and immunostaining confirmed the presence of HCN1-4 channel isoforms, likely arranged in different configurations, in LMCs. Hence, all together, data suggest that HCN channels might play an important role in affecting contraction frequency of LMCs.
The mechanism through which the stresses developed in the diaphragmatic tissue during skeletal muscle contraction sustain local lymphatic function was studied in 10 deeply anesthetized, tracheotomized adult Wistar rats whose diaphragm was exposed after thoracotomy. To evaluate the direct effect of skeletal muscle contraction on the hydraulic intraluminal lymphatic pressures (Plymph) and lymphatic vessel geometry, the maximal contraction of diaphragmatic fibers adjacent to a lymphatic vessel was elicited by injection of 9.2 nl of 1 M KCl solution among diaphragmatic fibers while Plymph was recorded through micropuncture and vessel geometry via stereomicroscopy video recording. In lymphatics oriented perpendicularly to the longitudinal axis of muscle fibers and located at <300 μm from KCl injection, vessel diameter at maximal skeletal muscle contraction (Dmc) decreased to 61.3 ± 1.4% of the precontraction value [resting diameter (Drest)]; however, if injection was at >900 μm from the vessel, Dmc enlarged to 131.1 ± 2.3% of Drest. In vessels parallel to muscle fibers, Dmc increased to 122.8 ± 2.9% of Drest. During contraction, Plymph decreased as much as 22.5 ± 2.6 cmH2O in all submesothelial superficial vessels, whereas it increased by 10.7 ± 5.1 cmH2O in deeper vessels running perpendicular to contracting muscle fibers. Hence, the three-dimensional arrangement of the diaphragmatic lymphatic network seems to be finalized to efficiently exploit the stresses exerted by muscle fibers during the contracting inspiratory phase to promote lymph formation in superficial submesothelial lymphatics and its further propulsion in deeper intramuscular vessels.
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