The influence of experimentally induced pathological states on the flow and composition of central lymph in the rat

The flow of central lymph in the rat was examined and its concentration of total proteins was determined. Experiments were performed both on healthy animals and on animals with experimentally induced pathological states. The following results were obtained: The flow of the lymph is increased in chronic liver damage, acute kidney damage and the malabsorption syndrome. On the other hand, lymph flow is decreased in fasting animals, and it is unaffected by acute liver damage. Total protein concentration was increased in fasting rats and in the group with acute liver damage, and on the contrary, it was decreased in the group with chronic damage, acute kidney damage, and malabsorption syndrome. The lymph flow or total protein concentration are not sex-dependent.     

Intrathoracic sources of caudal mediastinal lymph node efferent lymph in sheep

We investigated the intrathoracic contributions to the caudal mediastinal lymph node (CMN) efferent lymph in 12 anesthetized sheep after removing possible systemic contributions from below the diaphragm. We interrupted various pathways that may send lymph to the CMN (chest wall, esophagus, lung). Because the experiment is destructive, we did the resections in various combinations and waited 1 h between steps. Base-line CMN efferent lymph flow averaged 0.90 +/- 0.52 g/15 min (mean +/- SD). Cutting the pulmonary ligaments bilaterally caused lymph flow to decrease by an average of 58%. In five sheep, cauterizing around the lung hila reduced lymph flow by 16% of base line, cauterizing along the esophagus reduced lymph flow by 18% of base line, and cauterizing along the chest wall increased lymph flow by 6% of base line. After complete isolation of the node, except for the bronchoesophageal artery, dorsal mediastinal vein, and CMN efferent duct, 14% of base-line flow remained. The lymph-to-plasma total protein concentration ratios did not change significantly with any procedure. Under the conditions of our experiments, approximately 74% of base-line intrathoracic CMN efferent flow comes from the lung.     

Enhanced intestinal lymph formation during fat absorption: the importance of triglyceride hydrolysis.

The effect of intraduodenal administration of fats was studied in the rat to define the mechanisms responsible for the substantial increase in intestinal lymph flow and protein transport which follows fat ingestion. Triglyceride in the intestinal lumen, protected from hydrolysis, does not appear to enhance intestinal lymph production. Giving both long- and medium-chain fatty acids, however, causes intestinal lymph flow and protein transport to increase in a manner similar to that found after giving triglyceride which is allowed to undergo hydrolysis. Bile by itself does not seem to be responsible for the phenomenon.     

Effect of endotoxin on diaphragm lymph contamination in unanesthetized sheep

The preparation for collecting lung lymph from sheep caudal mediastinal lymph node (CMN) efferent vessels is widely used to study the effects of endotoxin on lung microvascular permeability. However, there are nonpulmonary lymph vessels that drain into the CMN along with the afferent lymph vessels from the lung. Thus CMN lymph is a mixture of lymph from the lung and diaphragm lymph vessels as well as from other nonpulmonary lymph vessels. We studied the effect of 0.5-1.0 microgram/kg Escherichia coli endotoxin on the flow rates in diaphragm and CMN efferent lymph vessels (Qdi and QCMN, respectively) in unanesthetized sheep. For the time period between 2 and 5.5 h after endotoxin QCMN was increased from its base line of 7.2 +/- 4.4 (SD) to 17.3 +/- 10.6 ml/h and the lymph-to-plasma protein concentration ratio (L/PCMN) had increased from 0.68 +/- 0.11 to 0.81 +/- 0.06. During the same time period, Qdi was 4.5 +/- 3.1 ml/h compared with 1.0 +/- 0.8 ml/h at base line and the diaphragm lymph-to-plasma protein concentration ratio (L/Pdi) was 0.92 +/- 0.07 (base line = 0.74 +/- 0.15). The increases in flow rate and protein concentration were significant for each type of vessel (P less than 0.05). We conclude that the period of increased QCMN and L/PCMN after endotoxin is associated with an increase in Qdi and L/Pdi. Thus, it is difficult to determine how much of the CMN lymph response comes from the lungs and how much comes from diaphragm lymph vessels.     

Comparison of flow rates and composition of ovarian lymph and blood in the day-16 pregnant rat

Rats (5) at Day 16 of pregnancy were anaesthetized and a modification of a venous outflow technique was used to collect ovarian venous blood and lymph for 2 h. Both fluids were analysed for progesterone, 20 alpha-dihydroprogesterone, total protein, transferrin and albumin concentrations. In addition SDS gel electrophoresis was carried out to obtain an initial indication of permeability of capillaries to the various protein fractions. The concentrations of progesterone and 20 alpha-dihydroprogesterone in ovarian lymph were only 37% and 48% respectively of the corresponding concentrations in the venous plasma. Total protein concentration in the lymph was 53% of the venous plasma. The albumin and transferrin concentrations were similarly lower in lymph than plasma but the difference was only significant for transferrin. This study confirms that the rate of lymph flow, per unit mass of tissue, is high for the ovary and represents about 1.1% of plasma flow. It shows also that of the total progestagens secreted only around 0.5% leave by the lymphatic route. The finding of relatively low progestagen concentrations in lymph questions the view that progestagens are transported by simple diffusion from the luteal cell to blood and raises the possibility of a counter-current flow between fluid in the interstitial space and blood.     

Effects of histamine and acetylcholine on equine digital lymph flow and composition.

We measured the flow rate and protein concentration of lymph collected from a digital lymphatic in eight anesthetized ponies. Additionally, we recorded systemic arterial pressure (Part), and small vein pressure (Psv). Control lymph flow averaged 0.068 ml/min, and contained 3.11 g/100 ml of protein with albumin/globulin ratio of 0.75. Twenty-minute local intra-arterial infusion of acetylcholine (10 mug/min.) elevated Psv but did not increase lymph flow rate or protein concentration. A 60-min local intra-arterial infusion of histamine (10 mug/min) produced a marked sustained increase in Psv and both lymph flow and protein concentration. Edema developed in the digit receiving histamine. These data support the conclusion that in the horse, as in other species, histamine edema is due primarily to a decreased transcapillary colloid osmotic pressure gradient rather than an increased transcapillary hydrostatic pressure gradient.     

Comparison of afferent and efferent lung lymph in the sheep

Efferent lymph collected from the caudal mediastinal lymph node (CMN) in the sheep lung lymph fistula model has been reported to represent free pulmonary interstitial fluid. Studies that utilize this model assume that nodal transit does not alter the composition of lymph. We collected afferent lymph from the tracheobronchial node (TBN) while simultaneously collecting CMN efferent lymph in acutely prepared sheep. We compared afferent and efferent lymph protein concentrations (CA and CE) and changes in flow rates (QLA and QLE) during base line and periods of elevated left atrial pressure (Pla). As a result of elevated Pla, QLA and QLE increased and the afferent lymph-to-plasma protein concentration ratio (CA/Cp) and the efferent lymph-to-plasma protein concentration ratio (CE/Cp) fell. The CA/Cp was significantly lower than the CE/Cp during base line (0.67 vs. 0.80) and periods of elevated Pla (0.41 vs. 0.61). Although we cannot exclude regional permeability differences, the difference between CA/Cp and CE/Cp is most likely due to the concentration of lymph within the CMN. Our data suggest nodal modification of CA is correlated with the afferent lymph-to-plasma colloid osmotic pressure ratio (pi A/pi p) and further suggest that nodal alteration of lymph during elevated Pla is due to the influence of decreased pi A/pi p at the blood-to-lymph barrier. We conclude that afferent lymph is a more accurate representation of lung free interstitial fluid because collection of pulmonary afferent lymph obviates the complications introduced by the CMN. Studies utilizing efferent lymph may have overestimated lung microvascular permeability in the acute sheep preparation.     

Removal of abdominal sources of caudal mediastinal node lymph in anesthetized sheep

We investigated the conditions for and the quantity of abdominal contributions to caudal mediastinal node (CMN) efferent lymph in 15 anesthetized sheep with open thorax and lung lymph fistulas. Contractions of the diaphragm by phrenic nerve stimulation increased lymph flow by 29%. Adding large volumes (10 ml/kg) of 5% albumin solution to the peritoneal cavity increased lymph flow by 19 and 28% during diaphragm contraction. Tracer albumin specific activity in lymph indicated that about half the lymph flow increment came from the peritoneal liquid. Raising hepatic vein pressure 19 cmH2O increased CMN lymph flow 178%. Lymph protein concentration also increased markedly, from 0.72 to 0.91 of the plasma concentration. We devised a simple procedure to cauterize across the pleural surface of the diaphragm to destroy the aberrant lymphatics. This procedure markedly diminished both abdominal cavity and portal system contributions.     

Effects of hypoproteinemia on lung microvascular protein sieving and lung lymph flow

Experiments were conducted on five chronically instrumented unanesthetized sheep to determine the effects of sustained hypoproteinemia on lung fluid balance. Plasma total protein concentration was decreased from a control value of 6.17 +/- 0.019 to 3.97 +/- 0.17 g/dl (mean +/- SE) by acute plasmapheresis and maintained at this level by chronic thoracic lymph duct drainage. We measured pulmonary arterial pressure, left atrial pressure, aortic pressure, central venous pressure, cardiac output, oncotic pressures of both plasma and lung lymph, lung lymph flow rate, and lung lymph-to-plasma ratio of total proteins and six protein fractions for both control base-line conditions and hypoproteinemia base-line conditions. Moreover, we estimated the average osmotic reflection coefficient for total proteins and the solvent drag reflection coefficients for the six protein fractions during hypoproteinemia. Hypoproteinemia caused significant decreases in lung lymph total protein concentration, lung lymph-to-plasma total protein concentration ratio, and oncotic pressures of plasma and lung lymph. There were no significant alterations in the vascular pressures, lung lymph flow rate, cardiac output, or oncotic pressure gradient. The osmotic reflection coefficient for total proteins was found to be 0.900 +/- 0.004 for hypoproteinemia conditions, which is equal to that found in a previous investigation for sheep with a normal plasma protein concentration. Our results suggest that hypoproteinemia does not alter the lung filtration coefficient nor the reflection coefficients for plasma proteins. Possible explanations for the reported increase in the lung filtration coefficient during hypoproteinemia by other investigators are also made.     

Effects of increased ventilation on lung lymph flow in unanesthetized sheep

To determine the effect of an increase in spontaneous minute ventilation on lung fluid balance, we added external dead space to the breathing circuit of six tracheostomized, unanesthetized, spontaneously breathing sheep in which lung lymph fistulas had been created surgically. The addition of 120-180 ml of dead space caused minute ventilation to increase by 50-100% (secondary to increases in both tidal volume and frequency), without changing pulmonary arterial pressure, pulmonary capillary wedge pressure, cardiac output, or arterial blood gas tensions. The increase in spontaneous ventilation was associated with an average increase of 27% in lung lymph flow (P less than 0.05) and an average reduction of 11% in the lymph-to-plasma concentration ratio (L/P) for total protein (P less than 0.05). Lymph flow and L/P for total protein approached stable values after 2-3 h of hyperpnea, and the increase in lymph flow persisted for at least 18 h of dead-space breathing. Removal of dead space was associated with a rapid return (within 45 min) of lymph flow to base-line levels. These results suggest that hyperpnea increases the pulmonary transvascular filtration rate. Since no changes in vascular pressures or cardiac output were observed, this increase in transvascular filtration is most likely due to a fall in interstitial fluid pressure.     

Serum and lymph lipids in rabbits with carbon tetrachloride-induced cirrhosis of the liver

Lymph flow and the composition of lymph lipids from the hepatic and thoracic ducts of rabbits with cirrhosis of the liver (induced by 46-51 intramuscular injections of a mixture of carbon tetrachloride and olive oil at 4-day intervals) have been compared with those of control animals injected with olive oil only. In cirrhotic animals, the concentration of lymph lipids was not greatly altered, but lymph flow, and consequently the hourly transport of lipids by lymph were greatly increased; the increase in transport of cholesteryl esters, free cholesterol, and phospholipids by way of the thoracic and hepatic duct lymph was particularly striking. The concentration of these lipid fractions in serum from the cirrhotic rabbits was also increased. The differences normally observed between lipid fatty acid compositions of serum and lymph disappeared in cirrhotic animals; this is interpreted as due to increased hepatic permeability to lipoproteins.     

Enzyme activities in thoracic duct lymph and plasma of anaesthetized, conscious resting and exercising dogs

The significance of changes in lymph flow for the extracellular distribution and transport of cellular enzymes and for the level of enzyme activities in plasma was investigated. Specimens of thoracic duct lymph were obtained from an extracorporal lymph shunt in anaesthetized, conscious resting and treadmill exercising dogs (6 km X h-1 for 1 h) The activity of 10 enzymes and of protein content in lymph and plasma were studied, as well as lymph flow, lymphatic transport, and the lymph-plasma ratio of these compounds. Lactate, pH, and blood gases were monitored in venous blood. Lymph flow of 0.80 ml X min-1 in anaesthetized dogs more than doubled (to 1.86 ml X min-1) when the animals were conscious and resting. In anaesthetized dogs lymph enzyme activity was higher only for enzymes of predominately hepatic origin, such as choline esterase (CHE) and alanine aminoferase (ALAT), and was lower for aspartate aminotransferase (ASAT) and aldolase (ALD). In conscious dogs, due to activation of the skeletal muscle "tissue pump", lymphatic transport of enzymes with rather high activity in skeletal muscle, and of protein, is significantly enhanced. Enzyme activities in plasma, however, did not differ between the groups. Lymph-plasma activity ratios higher than one were found for lactate dehydrogenase (LDH), malate dehydrogenase (MDH), ASAT, creatine kinase (CK), ALD, and phosphohexose isomerase (PHI). Exercise stimulated lymph flow up to 4.9 ml X min-1, and increased the lymphatic activities of those enzymes with a lymph-plasma ratio higher than unity, these enzymes increasing in the plasma due to the highly increased lymphatic transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of interstitial edema on lung lymph flow in goats in the absence of filtration.

We tested the effect of interstitial edema on lung lymph flow when no filtration occurred. In 16 anesthetized open-thorax ventilated supine goats, we set pulmonary arterial and left atrial pressures to nearly zero and measured lymph flow for 3 h from six lungs without edema and ten with edema. Lymph flow decreased exponentially in all experiments as soon as filtration ceased. In the normal lungs the mean half time of the lymph flow decrease was 12.7 +/- 4.8 (SD) min, which was significantly shorter (P less than 0.05) than the 29.1 +/- 14.8 min half time in the edematous lungs. When ventilation was stopped, lymph flow in the edematous lungs decreased as rapidly as in the normal lungs. The total quantity of lymph after filtration ceased was 2.7 +/- 0.8 ml in normal lungs and 9.5 +/- 6.3 ml in edematous lungs, even though extravascular lung water was doubled in the latter (8.4 +/- 2.4 vs. 3.3 +/- 0.4 g/g dry lung, P less than 0.01). Thus the maximum possible clearance of the interstitial edema liquid by the lymphatics was 6.3 +/- 4.8%. When we restarted pulmonary blood flow after 1-2 h in four additional goats, lymph flow recovered within 30 min to the baseline level. These findings support the hypothesis that lung lymph flow originates mainly from alveolar wall perimicrovascular interstitial liquid and that the contribution of the lung lymphatic system to the clearance of interstitial edema (bronchovascular cuffs, interlobular septa) is small.     

The effects of cholecystokinin and cholecystokinin-octapeptide on intestinal lymph flow in the rat

Intravenous cholecystokinin and its synthetic C-terminal octapeptide were found to cause a transient augmentation of intestinal lymph flow in the rat. Concomitant increase in lymph protein transport suggests that this reflects the increase in intestinal blood flow which is known to occur in response to these agents.     

Lung lymph flow during volume infusions

We investigated the effect of intravenous isotonic crystalloid solution infusion on lung lymph flow. Tracheobronchial lung lymph vessels were cannulated in 13 anesthetized dogs. The lymph flow rate was measured 1) with the lymph flowing against atmospheric pressure (QL), and 2) with the pressure at the outflow end of the lymph cannula equal to systemic venous pressure (QLV). QL and QLV were measured alternately in each lymph vessel. In one group of nine dogs, the base-line QL and QLV were 18 +/- 9 and 13 +/- 6 (SD) microliter/min, respectively (P less than 0.05). QL increased by 4.8 +/- 1.4-fold, and QLV increased by 3.5 +/- 2.1-fold during a 4-h infusion of 25 ml X kg-1 X h-1 of Ringer solution. QLV was significantly less than QL at all times. The increases in lymph flow were caused primarily by a reduction in the effective resistance of the lymph vessels with little rise in the pressure driving lymph from the lungs. Because QLV flowed against systemic venous pressure, the increase in QLV was blunted by a 3.1 +/- 2.3 cmH2O rise in venous pressure during the infusions. In the remaining four dogs, we infused Ringer solution rapidly in order to raise venous pressure to greater than 15 cmH2O. This caused QL to increase by 25 +/- 7-fold; however, QLV decreased to zero. We conclude that elevations in venous pressure which occur during volume infusions oppose lung lymph flow and lead to accumulation of excess fluid in the lungs.     

Effect of outflow pressure on lung lymph flow in unanesthetized sheep

Studies in anesthetized animals have shown that the flow rate from lung lymphatics (QL) depends on the pressure at the outflow end of the vessels (Po). We tested this in unanesthetized sheep prepared with chronic lung lymph cannula. We measured QL with the lymph cannula held at various heights above the olecranon and calculated Po as the height + QL X cannula resistance. QL decreased with increases in Po (delta QL/delta Po = -8.2 +/- 6.4 microliter X min-1 X cmH2O-1, mean +/- SD). We increased QL by raising left atrial pressure or infusing Ringer solution or Escherichia coli endotoxin and found that QL was even more sensitive to Po (delta QL/delta Po = -32 +/- 22). Cannula resistance caused a 9-70% reduction in QL. Changes in QL caused by increasing Po were not associated with changes in lymph protein concentration for up to 330 min. This indicates that increases in Po shunt lymph away from cannulated vessels but do not substantially effect microvascular filtration rate. The shunted lymph may flow into other vessels or collect in the lung. We conclude that QL does not accurately represent microvascular filtration rate because it depends on the cannula resistance and position at which the investigator chooses to place the cannula.     

Oscillatory processes in lymph microcirculation in the human skin

This study was the first to use laser Doppler flowmetry followed by wavelet analysis in order to estimate oscillations in lymph microcirculation in 30 subjects with (n = 13) or without (n = 17) edema of the distal part of the upper limb. Lymph flow in the human skin exhibited clear dominance of pacemaker phase oscillations in the frequency ranges of 0.021–0.042 and 0.016–0.035 Hz in the skin of the palm surface of the finger nail bone and in the skin of the forearm, respectively. Edema was associated with an increase in the peak frequencies and normalized maximum amplitudes (Al/Ml, where Al is the mean value of the maximum amplitude of phase oscillations, and Ml is the value of the averaged lymph flow expressed in perfusion units). Low-amplitude oscillations were recorded rarer in the myogenic, endothelial, and respiratory ranges. We did not find any cardiac pulse rhythm in the wavelet spectrum of the lymph flow. We did not find any interaction between the Al/Ml value and the skin temperature. In the group of subjects without edema, under physiological conditions only, we found a negative correlation between the Al/Ml value and the amplitudes of myogenous proper blood flow oscillations, which reflected the number of functional capillaries and activity of oxidative metabolism in the tissue. In the group with edema, we did not find any correlations between the indices of lymph flow and blood flow. The values of normalized amplitude and frequency of phase oscillations may be used as efficient diagnostic tools in the studies on lymph microcirculation.     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

Contamination of lung lymph following standard and modified procedures in sheep

The sheep lung lymph fistula preparation of Staub et al. is reported to be contaminated by systemic lymph. The published estimates of contamination range from 5% (awake sheep) to 60% (anesthetized sheep). In view of these conflicting estimates, we investigated the pre- and postoperative contaminating sources, morphological and functional consequences of the proposed contamination reducing modifications, and base-line lung lymph flow in awake sheep following standard and modified cannulation procedures. Our morphological observations are not compatible with the higher estimates of contamination (25-60%). Evidence of lymph leakage from cauterized lymphatics was found. The lymphatics that appear after diaphragmatic cautery and partial resection of caudal mediastinal lymph node were found to constitute "new" contaminating sources. The lymph flow data from base-line and increased vascular pressure conditions were consistent with the reported low estimates of contamination (5%). We propose simple modifications of the standard procedure of Staub et al. which may be nearly as effective in reducing contamination by extrapulmonary lymph as the more invasive and/or traumatic modifications.     

Estimation of equivalent pore radii in pulmonary microvasculature after lung lymph fistula preparation

The effect of lung lymph fistula preparation on pulmonary microvascular permeability was investigated in sheep. Acutely prepared animals (n = 9) were compared with animals with a chronic lung lymph fistula (n = 5). The osmotic reflection coefficients (sigma) for total protein, albumin, immunoglobins (Ig) G and M, and the equivalent pore dimensions were calculated. Data were achieved at maximal possible lymph flows (QL) following elevation of left atrial pressure. In sheep with a chronic lung lymph fistula sigma's for total protein, albumin, IgG, and IgM at maximal lymph flows were 0.76 +/- 0.01, 0.65 +/- 0.09, 0.79 +/- 0.03, and 0.91 +/- 0.01, respectively. In the acutely prepared group the minimum lymph-to-plasma protein concentration for total protein was 0.39 +/- 0.06, corresponding to a sigma of 0.61 +/- 0.01. The sigma for albumin, IgG, and IgM were 0.48 +/- 0.04, 0.64 +/- 0.02, and 0.87 +/- 0.01, respectively. The equivalent pore radii in the chronic group were determined to be 54 and 190 A with 29% of the filtration accounted for by large pores. In the acute group the small pores were 56 A and the large pores 175 A with 53% of total volume flow at maximum lymph flows occurring through the large pores. Assuming a constant small-pore population the large pore number increased 4.5 times after surgery. For total protein, IgG, and IgM, sigma's in the acutely prepared group were significantly lower than in the control group. These results thus indicate that surgical preparation of a lung lymph fistula in sheep may cause acute increases in pulmonary microvascular permeability.