Fluid Osmolarity Modulates the Rate of Spontaneous Contraction of Lymphatic Vessels and Lymph Flow by Means of a Cooperation between TRPV and VRAC Channels.

Lymphatic vessels are capable of sustaining lymph formation and propulsion via an intrinsic mechanism based on the spontaneous contraction of the lymphatic muscle in the wall of lymphatic collectors. Exposure to a hyper- or hypo-osmolar environment can deeply affect the intrinsic contraction rate and therefore alter lymph flow. In this work, we aimed at defining the putative receptors underlying such a response. Functional experiments were conducted in ex vivo rat diaphragmatic specimens containing spontaneously contracting lymphatic vessels that were exposed to either hyper- or hypo-osmolar solutions. Lymphatics were challenged with blockers to TRPV4, TRPV1, and VRAC channels, known to respond to changes in osmolarity and/or cell swelling and expressed by lymphatic vessels. Results show that the normal response to a hyperosmolar environment is a steady decrease in the contraction rate and lymph flow and can be prevented by blocking TRPV1 channels with capsazepine. The response to a hyposmolar environment consists of an early phase of an increase in the contraction rate, followed by a decrease. The early phase is abolished by blocking VRACs with DCPIB, while blocking TRPV4 mainly resulted in a delay of the early response. Overall, our data suggest that the cooperation of the three channels can shape the response of lymphatic vessels in terms of contraction frequency and lymph flow, with a prominent role of TRPV1 and VRACs.

Draining the Pleural Space: Lymphatic Vessels Facing the Most Challenging Task.

Lymphatic vessels exploit the mechanical stresses of their surroundings together with intrinsic rhythmic contractions to drain lymph from interstitial spaces and serosal cavities to eventually empty into the blood venous stream. This task is more difficult when the liquid to be drained has a very subatmospheric pressure, as it occurs in the pleural cavity. This peculiar space must maintain a very low fluid volume at negative hydraulic pressure in order to guarantee a proper mechanical coupling between the chest wall and lungs. To better understand the potential for liquid drainage, the key parameter to be considered is the difference in hydraulic pressure between the pleural space and the lymphatic lumen. In this review we collected old and new findings from in vivo direct measurements of hydraulic pressures in anaesthetized animals with the aim to better frame the complex physiology of diaphragmatic and intercostal lymphatics which drain liquid from the pleural cavity.

Interplay between Gut Lymphatic Vessels and Microbiota.

Lymphatic vessels play a distinctive role in draining fluid, molecules and even cells from interstitial and serosal spaces back to the blood circulation. Lymph vessels of the gut, and especially those located in the villi (called lacteals), not only serve this primary function, but are also responsible for the transport of lipid moieties absorbed by the intestinal mucosa and serve as a second line of defence against possible bacterial infections. Here, we briefly review the current knowledge of the general mechanisms allowing lymph drainage and propulsion and will focus on the most recent findings on the mutual relationship between lacteals and intestinal microbiota.

Lymphatic Vessels and Their Surroundings: How Local Physical Factors Affect Lymph Flow.

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.

Paracrine effect of human adipose-derived stem cells on lymphatic endothelial cells.

Aim: The proposal of this study was to evaluate, in vitro, the potential paracrine effect of human adipose-derived stem cells (hASCs) to promote lymphangiogenesis in lymphatic endothelial cells isolated from rat diaphragmatic lymphatic vessels. Materials & methods: ELISA on VEGFA, VEGFC and IL6 in hASC-conditioned medium; LYVE1 immunostaining; and gene expression of PROX1, VEGFR3, VEGFC, VEGFA and IL6 were the methods used. Results: In 2D culture, hASC-conditioned medium was able to promote lymphatic endothelial cell survival, maintenance of endothelial cobblestone morphology and induction to form a vessel-like structure. Conclusion: The authors' results represent in vitro evidence of the paracrine effect of hASCs on lymphatic endothelial cells, suggesting the possible role of hASC-conditioned medium in developing new therapeutic approaches for lymphatic system-related dysfunction such as secondary lymphedema.

TRPV4 channels' dominant role in the temperature modulation of intrinsic contractility and lymph flow of rat diaphragmatic lymphatics.

The lymphatic system drains and propels lymph by extrinsic and intrinsic mechanisms. Intrinsic propulsion depends upon spontaneous rhythmic contractions of lymphatic muscles in the vessel walls and is critically affected by changes in the surrounding tissue like osmolarity and temperature. Lymphatics of the diaphragm display a steep change in contraction frequency in response to changes in temperature, and this, in turn, affects lymph flow. In the present work, we demonstrated in an ex vivo diaphragmatic tissue rat model that diaphragmatic lymphatics express transient receptor potential channels of the vanilloid 4 subfamily (TRPV4) and that their blockade by both the nonselective antagonist Ruthenium Red and the selective antagonist HC-067047 abolished the response of lymphatics to temperature changes. Moreover, the selective activation of TRPV4 channels by means of GSK1016790A mirrored the behavior of vessels exposed to increasing temperatures, pointing out the critical role played by these channels in sensing the temperature of the lymphatic vessels' environment and thus inducing a change in contraction frequency and lymph flow.NEW & NOTEWORTHY The present work addresses the putative receptor system that enables diaphragmatic lymphatics to change intrinsic contraction frequency and thus lymph flow according to the changes in temperature of the surrounding environment, showing that this role can be sustained by TRPV4 channels alone.

Acute Exposure of Collecting Lymphatic Vessels to Low-Density Lipoproteins Increases Both Contraction Frequency and Lymph Flow: An In Vivo Mechanical Insight.

Background: Lymphatic vessels drain fluids and solutes from interstitial spaces and serosal cavities. Among the solutes, low-density lipoproteins (LDL) are drained and can be detected in peripheral lymph, where they have been reported to exert a modulatory action on lymphatic vessels intrinsic contraction rate. In the present work, we investigated lymphatic vessel mechanical properties (contraction frequency and amplitude) that may be modulated by LDL application and the consequence on lymph flow. Methods and Results: Human-derived LDL were resuspended in phosphate-buffered saline (PBS) and microinjected in the interstitial space surrounding spontaneously contracting lymphatic vessels of the rat diaphragm, in vivo. Vessels' contraction rate and diameter were measured in control conditions (PBS) and after LDL injection. Lymph flow (Jlymph) was computed from contraction rate and diameter change. In some animals, after the recording procedure, diaphragmatic tissue samples were excised and immunostained with antilymphatic muscle (LM) actin to investigate the correlation between LM signal level and contraction amplitude. Data indicate a positive, saturating correlation between the abundance of LM actin and contraction amplitude, and LDL microinjection caused an acute increase in contraction frequency (+126%), a reduction of contraction amplitude to 75% of that obtained after PBS injection, and a +63% increase in Jlymph. Conclusions: From our in vivo analysis of the mechanical parameters affected by LDL, Jlymph was increased by a predominant effect on the contraction rate rather than amplitude, suggesting that the still elusive messaging system might be linked to the pacemaker sites.

Method for Studying ECM Expression: In Situ RT-PCR.

The gene expression of the extracellular matrix macromolecules is critical in the analysis of various pathologies. The use of a RT-PCR directly on a fixed tissue enables the recognition of the real expressing cells for any ECM molecules together with the tissue localization. The method here described is easy to perform using the same material as for common immunostaining and the same primers used for quantitative RT-PCR. Moreover, the used primers, designed with a final amplicon that spans the exon-exon junction, allow to detect the cDNA but not the gDNA sequences.

Fluid Osmolarity Acutely and Differentially Modulates Lymphatic Vessels Intrinsic Contractions and Lymph Flow.

Lymph formation and propulsion rely on an extrinsic mechanism based on the forces that surrounding tissues exert upon the vessel wall and lumen and an intrinsic mechanism based on spontaneous, rhythmic contractions of the lymphatic muscle layer of collecting vessels. The two spontaneous pacemakers described in literature involve chloride-dependent depolarizations (STDs) and If-like currents, both giving rise to a variable contraction frequency (fc) of lymphatic vessels functional units (lymphangions). Several stimuli have been shown to modulate fc, such as temperature, shear stress, and several tissue chemical modulators (prostaglandins, norepinephrine, acetylcholine, substance P, and others). However, no detailed description is present in literature on the acute modulation of fc by means of osmolarity change of the surrounding interstitial space. Using a well-developed ex-vivo rat diaphragmatic preparation, in which osmolarity was changed by varying the concentration of D-mannitol in the perfusing solution and in later experiments the concentration of NaCl and then of Na+ and Cl- ions separately by ionic substitution, we provide detailed experimental evidences that a stepwise increase in osmolarity from control value (308 mOsm) up to 324 mOsm caused a reduction of fc down to ~-70% within the first 14 min, and that a stepwise decrease in osmolarity up to 290 mOsm induced an early fc increase to ~+34% of control, followed by a decline to an fc of ~-18% of control value. These variations were more dramatic when the same osmolarity changes were obtained by varying NaCl and/or Na+ or Cl- ions concentration, which caused an almost complete arrest of spontaneous contractility within 14 min from the application. Diastolic and systolic diameters and stroke volume were not affected by osmolarity changes, so that modulation of lymph flow closely followed that of fc. Modulation of lymph flow secondary to osmolarity changes is relevant if one considers that interstitial fluid balance is also dependent upon lymph drainage, and thus it is possible that, at least in the acute phase following variations of interstitial fluid osmolarity, its volume control might eventually be impaired due to the reduced or in the worst scenario null lymph drainage.

Temperature-dependent modulation of regional lymphatic contraction frequency and flow.

Lymph drainage and propulsion are sustained by an extrinsic mechanism, based on mechanical forces acting from the surrounding tissues against the wall of lymphatic vessels, and by an intrinsic mechanism attributable to active spontaneous contractions of the lymphatic vessel muscle. Despite being heterogeneous, the mechanisms underlying the generation of spontaneous contractions share a common biochemical nature and are thus modulated by temperature. In this study, we challenged excised tissues from rat diaphragm and hindpaw, endowed with spontaneously contracting lymphatic vessels, to temperatures from 24°C (hindpaw) or 33°C (diaphragmatic vessels) to 40°C while measuring lymphatic contraction frequency (fc) and amplitude. Both vessel populations displayed a sigmoidal relationship between fc and temperature, each centered around the average temperature of surrounding tissue (36.7 diaphragmatic and 32.1 hindpaw lymphatics). Although the slope factor of the sigmoidal fit to the fc change of hindpaw vessels was 2.3°C·cycles-1·min-1, a value within the normal range displayed by simple biochemical reactions, the slope factor of the diaphragmatic lymphatics was 0.62°C·cycles-1·min-1, suggesting the added involvement of temperature-sensing mechanisms. Lymph flow calculated as a function of temperature confirmed the relationship observed on fc data alone and showed that none of the two lymphatic vessel populations would be able to adapt to the optimal working temperature of the other tissue district. This poses a novel question whether lymphatic vessels might not adapt their function to accommodate the change if exposed to a surrounding temperature, which is different from their normal condition.NEW & NOTEWORTHY This study demonstrates to what extent lymphatic vessel intrinsic contractility and lymph flow are modulated by temperature and that this modulation is dependent on the body district that the vessels belong to, suggesting a possible functional misbehavior should lymphatic vessels be exposed to a chronically different temperature.

Hyperpolarization-activated cyclic nucleotide-gated channels in peripheral diaphragmatic lymphatics.

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 (FL) and increased end-diastolic diameter (DL-d, +7.3%, P < 0.01) without changes in end-systolic diameter (DL-s). Ivabradine (300 µM) abolished contraction and increased DL-d (-14%, n = 10, P < 0.01) or caused an incomplete inhibition of FL (n = 3, P < 0.01), leaving DL-d and DL-s unaltered. ZD-7288 (200 µM) completely (n = 12, P < 0.01) abolished FL, while DL-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.

Lymph flow pattern in pleural diaphragmatic lymphatics during intrinsic and extrinsic isotonic contraction.

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 ± 2.9 µm at an average net forward speed of 18.0 ± 3.6 µm/s. Each skeletal muscle contraction sustained a net forward-lymph displacement of 441.9 ± 159.2 µm at an average velocity of 339.9 ± 122.7 µm/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.

Regional lung tissue changes with mechanical ventilation and fluid load.

PURPOSE: To investigate the regional gravity-dependent impact of mechanical ventilation and fluid overload on lung extracellular matrix (ECM) in healthy lungs. MATERIALS AND METHODS: The glycosaminoglycans (GAGs) composition of the ventral and dorsal lung parenchyma was determined in anesthetized supine healthy rats mechanically ventilated for 4 hours in air: (a) at low (∼7.5 mL/kg) or high (∼ 23 mL /kg) tidal volume (V(T)) and 0 cmH2O positive end-expiratory pressure (PEEP); (b) at low or high V(T) at 5 cmH2O PEEP and (c) with or without 7 mL /(kg·h) intravenous saline infusion. RESULTS: Mechanical ventilation degraded lung ECM, with alveolar septa thinning and structural GAGs disorganization. Low V(T) ventilation was associated with significant tissue structure changes in both ventral and dorsal lung regions, while high VT mainly affected the dependent ones. PEEP decreased ECM injury mainly in the ventral lung regions, although it did not prevent matrix fragmentation and washout at high V(T). Intravascular fluid load increased lung damage prevalently in the ventral lung regions. CONCLUSION: Mechanical ventilation and fluid load may cause additive injuries in healthy lungs, mainly in ventral regions.

Diaphragmatic lymphatic vessel behavior during local skeletal muscle contraction.

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.

Spontaneous activity in peripheral diaphragmatic lymphatic loops.

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 ∼0.35 s between active and stretch-activated and 0.54 s between stretch-activated and passive segments, promoting a uniform lymph flux of ∼150/200 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.

Impact of mechanical ventilation and fluid load on pulmonary glycosaminoglycans.

The combined effect of mechanical ventilation and fluid load on pulmonary glycasaminoglycans (GAGs) was studied in anaesthetized rats ((BW 290±21.8 (SE)g) mechanically ventilated for 4h: (a) at low (∼7.5mlkg(-1)) or high (∼23mlkg(-1)) tidal volume (V(T)) and zero alveolar pressure; (b) at low or high V(T) at 5cmH(2)O positive end-expiratory pressure (PEEP); (c) with or without 7mlkg(-1)h(-1) intravenous infusion of Phosphate Buffer Solution (PBS). Compared to spontaneous breathing, GAGs extractability decreased by 52.1±1.5% and 42.2±7.3% in not-infused lungs mechanically ventilated at low V(T) or at high V(T) and PEEP, respectively. In contrast, in infused lungs, GAGs extractability increased by 56.1±4.0% in spontaneous ventilation and PEEP and up to 81.1% in all mechanically ventilated lungs, except at low V(T) without PEEP. In the absence of an inflammatory process, these results suggest that PEEP was protective at low but not at high V(T) when alveolar structures experience exceedingly high stresses. When combined to mechanical ventilation, fluid load might exacerbate edema development and lung injury.

Tissue contribution to the mechanical features of diaphragmatic initial lymphatics.

The role of the mechanical properties of the initial lymphatic wall and of the surrounding tissue in supporting lymph formation and/or progression was studied in six anaesthetized, neuromuscularly blocked and mechanically ventilated rats. After mid-sternal thoracotomy, submesothelial initial lymphatics were identified on the pleural diaphragmatic surface through stereomicroscopy. An 'in vivo' lymphatic segment was prepared by securing two surgical threads around the vessel at a distance of ∼2.5 mm leaving the vessel in place. Two glass micropipettes were inserted into the lumen, one for intraluminar injections of 4.6 nl saline boluses and one for hydraulic pressure (Plymph) recording. The compliance of the vessel wall (Clymph) was calculated as the slope of the plot describing the change in segment volume as a function of the post-injection Plymph changes. Two superficial lymphatic vessel populations with a significantly different Clymph (6.7 ± 1.6 and 1.5 ± 0.4 nl mmHg−1 (mean ± S.E.M.), P < 0.001) were identified. In seven additional rats, the average elastic modulus of diaphragmatic tissue strips was determined by uniaxial tension tests to be 1.7 ± 0.3 MPa. Clymph calculated for an initial lymphatic completely surrounded by isotropic tissue was 0.068 nl mmHg−1, i.e. two orders of magnitude lower than in submesothelial lymphatics. Modelling of stress distribution in the lymphatic wall suggests that compliant vessels may act as reservoirs accommodating large absorbed fluid volumes, while lymphatics with stiffer walls serve to propel fluid through the lumen of the lymphatic vessel by taking advantage of the more efficient mechanical transmission of tissue stresses to the lymphatic lumen.

Kinetics of fluid flux in the rat diaphragmatic submesothelial lymphatic lacunae.

The specific role of loops and/or linear segments in pleural diaphragmatic submesothelial lymphatics was investigated in seven anesthetized, paralyzed, and mechanically ventilated rats. Lymphatic loops lay peripherally above the diaphragmatic muscular plane, whereas linear vessels run over both the muscular and central tendineous regions. Lymph vessel diameter, measured by automatic software analysis, was significantly greater (P < 0.01) in linear vessels [103.4 +/- 8.5 microm (mean +/- SE), n = 18] than in loops (54.6 +/- 3.3 microm, n = 21). Conversely, the geometric mean of intraluminal flow velocity, obtained from the speed of distribution of a bolus of fluorescent dextrans injected into the vessel, was lower (P < 0.01) in linear vessels (26.3 +/- 1.4 microm/s) compared with loops (51.3 +/- 3.2 microm/s). Lymph flow, calculated as the product of flow velocity by vessel cross-sectional area, was similar in linear vessels and in individual vessels of a loop, averaging 8.6 +/- 1.6 nl/min. Flow was always directed from the diaphragm periphery toward the medial tendineous region in linear vessels, whereas it was more complex and evidently controlled by intraluminal unidirectional valves in loops. The results suggest that loops might be the preferential site of lymph formation, whereas linear vessels would be mainly involved in the progression of newly formed lymph toward deeper collecting diaphragmatic ducts. Within the same hierarchic order of diaphragmatic lymphatic vessels, the spatial organization and geometrical arrangement of the submesothelial lacunae seem to be finalized at exploiting the alternate contraction/relaxation phases of diaphragmatic muscle fibers to optimize fluid removal from serosal cavities.

Proteoglycan fragmentation and respiratory mechanics in mechanically ventilated healthy rats.

This research investigated whether stretching of lung tissue due to increased positive alveolar pressure swings during mechanical ventilation (MV) at various tidal volumes (V(T)) might affect the composition and/or structure of the glycosaminoglycan (GAG) components of pulmonary extracellular proteoglycans. Experiments were performed in 30 healthy rats: 1) anesthetized and immediately killed (controls, C-0); 2) anesthetized and spontaneously breathing for 4 h (C-4h); and 3) anesthetized, paralyzed, and mechanically ventilated for 4 h with air at 0-cmH(2)O end-expiratory pressure and V(T) of 8 ml/kg (MV-1), 16 ml/kg (MV-2), 24 ml/kg (MV-3), or 32 ml/kg (MV-4), adjusting respiratory rates at a minute ventilation of 270 ml/min. Compared with C-0 and C-4h, a significant reduction of dynamic and static compliance of the respiratory system and of the lung was observed only in MV-4, while extravascular lung water significantly increased in MV-3 and MV-4, but not in MV-1 and MV-2. However, even in MV-1, MV induced a significant fragmentation of pulmonary GAGs. Extraction of covalently bound GAGs and wash out of loosely bound or fragmented GAGs progressively increased with increasing V(T) and was associated with increased expression of local (matrix metalloproteinase-2) and systemic (matrix metalloproteinase-9) activated metalloproteases. We conclude that 1) MV, even at "physiological" low V(T), severely affects the pulmonary extracellular architecture, exposing the lung parenchyma to development of ventilator-induced lung injury; and 2) respiratory mechanics is not a reliable clinical tool for early detection of lung injury.

Regional recruitment of rat diaphragmatic lymphatics in response to increased pleural or peritoneal fluid load.

The specific role of the diaphragmatic tendinous and muscular tissues in sustaining lymph formation and propulsion in the diaphragm was studied in 24 anaesthetized spontaneously breathing supine rats. Three experimental protocols were used: (a) control; (b) peritoneal ascitis, induced through an intraperitoneal injection of 100 ml kg(-1) of iso-oncotic saline; and (c) pleural effusion, induced through an intrapleural injection of 6.6 ml kg(-1) saline solution. A group of animals (n = 12) was instrumented to measure the hydraulic transdiaphragmatic pressure gradient between the pleural and peritoneal cavities in the three protocols. In the other group (n = 12), the injected iso-oncotic saline was enriched with 2% fluorescent dextrans (molecular mass = 70 kDa); at 30 min from the injections these animals were suppressed and their diaphragm excised and processed for confocal microscopy analysis. In control conditions, in spite of a favourable peritoneal-to-pleural pressure gradient, the majority of the tracer absorbed into the diaphragmatic lymphatic system converges towards the deeper collecting lymphatic ducts. This suggests that diaphragmatic lymph formation mostly depends upon pressure gradients developing between the serosal cavities and the lymphatic vessel lumen. In addition, the tracer distributes to lymph vessels located in the muscular diaphragmatic tissue, suggesting that active muscle contraction, rather than passive tendon stretch, more efficiently enhances local diaphragmatic lymph flow. Vice versa, a prevailing recruitment of the lymphatics of the tendinous diaphragmatic regions was observed in peritoneal ascitis and pleural effusion, suggesting a functional adaptation of the diaphragmatic network to increased draining requirements.