PS products and capillary reflection coefficients from analysis of lymphatic protein flux data.
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Animals↗
Biomedical subjects
R K Reed
Publications and source records attributed to R K Reed.
Increased negativity of interstitial fluid pressure in rat trachea in dextran anaphylaxis.
This study shows that, in rat trachea, dextran anaphylaxis is associated with increased negativity of interstitial fluid pressure (Pif) as measured with sharpened glass capillaries (tip diameter 3-7 microns) connected to a servo-controlled counterpressure system. Experiments were carried out in pentobarbital-anesthetized Wistar-Møller rats. Pif in the control situation was -2.5 +/- 0.38 (SD) mmHg. The mean pressure in animals killed 2 min after initiation of the anaphylactic reaction by injection of 1 ml of 10% Dextran 70 in 0.9% NaCl was -10.3 +/- 2.6 mmHg. In another experimental series, interstitial fluid volume was measured after dextran administration but without inducing circulatory arrest. Interstitial fluid volume increased from 0.94 +/- 0.16 to 1.56 +/- 0.42 ml/g dry wt after 10 min to 1.57 +/- 0.30 and 1.10 +/- 0.27 ml/g dry wt after 30 and 60 min, respectively. The increased negativity in Pif in tracheal mucosa in the early phase of dextran anaphylaxis will markedly increase the transcapillary net filtration pressure in the initial phase of edema development.
Anaphylaxis↗
Turnover of hyaluronan in the rabbit pleural space.
Hyaluronan influences lung fluid balance. The clearance of lung hyaluronan by way of the pulmonary lymphatics and the pleural space is increased when fluid flux into the interstitium is increased. The purpose of this study was to determine the rate at which hyaluronan is removed from the pleural space. We injected hyaluronan, labeled with tritium in the acetyl group, into the pleural space of six rabbits. The appearance of [3H]H2O in serum was measured over time to calculate the turnover rate of hyaluronan. We found that the half-lives ranged between 8 and 15 h and were positively related to the amount of hyaluronan injected. At the end of the experiment, the contralateral pleural space was irrigated to determine the amount of pleural space hyaluronan, which was 0.3 micrograms/kg body wt.
Animals↗
Blockade of beta 1-integrins in skin causes edema through lowering of interstitial fluid pressure.
The increased capillary fluid filtration required to rapidly create edema in acute inflammation can be generated by increased negativity of the interstitial fluid pressure (Pif). This observation suggests that connective tissues can "actively" enhance capillary fluid filtration. We now show that in vivo blockade of beta 1-integrin adhesion receptors in rat skin causes local edema concomitant with increased negativity of Pif. Experiments were performed on the dorsal side of the hind paw, and Pif was measured with sharpened glass capillaries (tip diameter, 3-7 microns) connected to a servo-controlled counterpressure system. Measurements were made after circulatory arrest had been induced with intracardiac potassium chloride in pentobarbital anesthesia. This procedure prevents the vascular phenomena of increased fluid and protein flux leading to edema formation, which in turn can increase Pif and therefore potentially mask an increased negativity of Pif. Control Pif averaged -0.58 +/- 0.81 (mean +/- SD) mm Hg (n = 37). Subdermal injection of 5 microliters monospecific rabbit anti-rat integrin beta 1-subunit immunoglobulin G caused increased negativity of Pif to average values between -4 and -6 mm Hg within 10 minutes after injection. Subdermal injection of 0.9% NaCl, preimmune immunoglobulin G, rat anti-fibronectin, and peptides with Arg-Gly-Asp and Arg-Gly-Glu sequences did not change Pif significantly. In another series of experiments, 5 microliters anti-beta 1 integrin immunoglobulin G was injected subdermally in rats with intact circulation and resulted in an increase in total tissue water corresponding to a doubling of the interstitial fluid volume in 10 minutes (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
Turnover of hyaluronan in the microcirculation.
Hyaluronan in skin, lung, and intestine turns over within a few days and catabolism takes place locally in the tissues, in local lymph nodes, and in the liver. Hyaluronan will affect microcirculatory exchange through its influence on interstitial volume exclusion, hydraulic conductivity, and diffusivity of macromolecules. Prolonged increase in interstitial fluid flux in intestine has been shown to reduce the hyaluronan content, which in turn increases hydraulic conductivity and diffusivity of macromolecules.
Animals↗
Increased plasma concentrations of hyaluronan after major thermal injury in the rat.
Hyaluronan (HYA) is an ubiquitous polysaccharide in connective tissue interstitium; its normal plasma concentration is in the nanogram/ml range. Following major burn injury in sheep, plasma HYA can increase to levels tenfold greater than normal. The present study aimed to determine the effects of major cutaneous burns on plasma HYA concentration in rat, since in this species, the HYA elimination kinetics may better resemble those in man. Thermal injury did not alter the weight-average molecular weight of HYA in skin. HYA concentration in plasma was 46 +/- 4 ng/ml (mean +/- SEM) in controls. Three hours after scald burns to 40% of the body surface area, plasma HYA was 61 +/- 10 ng/ml in unresuscitated animals (P less than 0.1 vs. controls). After fluid replacement by lactated Ringer's plasma HYA was 75 +/- 8 ng/ml (P less than 0.01 vs. control), whereas plasma infusion prevented the elevation of plasma HYA. HYA also increased significantly after lactated Ringer's infusion in noninjured animals. The increased plasma concentration of HYA after major burns is probably a consequence of increased lymph flow, increasing its transport from skin interstitium. Possibly, plasma concentrations of connective tissue components may be used as indicators of the severity and extent of burn injuries.
Animals↗
Increased hyaluronan flux from skin following burn injury.
Hyaluronan (formerly hyaluronic acid) is an important constituent of the interstitial matrix in skin. Following major burn injury in animal models, plasma hyaluronan can increase to levels 10-fold greater than normal. The present experiments were designed to determine whether this is a result of the increased lymph flow (QL) accompanying the injury or of an increased release of hyaluronan from the burned skin and subcutaneous tissue. The lateral saphenous vein and a prenodal lymphatic were cannulated in the hindpaw of five anesthetized canines. Hindpaw venous pressure was elevated until the total protein concentration in lymph declined to steady-state levels, and QL and hyaluronan flux (QL X [hyaluronan]) was measured. A minor burn was inflicted by immersion of the paw into 100 degrees C water for five sec, and measurements were repeated at regular intervals for a minimum of 4 hr. Burn injury resulted in significant and persistent increases in QL (154 +/- 61 microliters/min versus 562 +/- 105 microliters/min 4 hr postburn) and lymph total protein concentration (1.34 +/- 0.04 g/dl versus 4.08 +/- 0.18 g/dl 4 hr postburn), while lymph hyaluronan concentration fell (3.01 +/- 0.20 micrograms/ml versus 2.1 +/- 0.16 micrograms/ml 4 hr postburn). The resultant increase in hyaluronan flux (0.42 +/- 0.13 microgram/min versus 1.17 +/- 0.22 microgram/min 4 hr postburn) appears to be a function of lymph flow rather than burn-induced release of skin hyaluronan. Hence, the increased plasma concentration of hyaluronan following major burns is likely a consequence of increased lymph flow from the site of injury.
Animals↗
Marked increase of plasma hyaluronan after major thermal injury and infusion therapy.
Hyaluronan (HYA) is an important structural element in skin and is presumably participating in regulation of the interstitial fluid volume. HYA is transported via the lymphatics from the tissues to the blood, where its concentration is normally very low. Fluid flux through the interstitium is markedly increased after thermal injury. The present study was performed to determine whether major thermal injury would affect plasma levels of HYA. In halothane-anesthetized sheep subjected to 40% BSA full-thickness scald burns, plasma HYA concentration increased from 116 +/- 19 (mean +/- SEM) to 172 +/- 18 ng/ml within 1 hr after injury (P less than 0.05). After 3 hr of fluid therapy plasma HYA concentration was further elevated to 10 times baseline (1417 +/- 322 ng/ml) (P less than 0.01). To clarify whether this rise represented an increased "washout" of interstitial HYA, attributable either to the burn injury or the subsequent fluid therapy, awake sheep were subjected to overhydration. Following a 3-hr infusion of lactated Ringer's 2.5 liter/hr, plasma HYA concentration increased to 2-3 times baseline. Lung lymph flow and its concentration of HYA increased, leading to an increase in the lymphatic flux of HYA to 10-20 times baseline. In peripheral lymph HYA flux increased 2-3 times baseline. Infusion of lactated Ringer's markedly increased lymphatic removal of HYA. However, plasma concentrations of HYA were 3 times higher after thermal injury than following fluid challenge alone, suggesting that thermal injury per se may also increase input of HYA into the systemic circulation.
Animals↗
Thermal skin injury: effect of fluid therapy on the transcapillary colloid osmotic gradient.
The effects of fluid therapy on interstitial colloid osmotic and hydrostatic pressures in thermally injured skin were investigated in anesthetized rats subjected to full-thickness scald burns to 40% of the body surface area and resuscitation for 3 hr by either lactated Ringer's or plasma. Interstitial fluid hydrostatic pressure (Pif) was reduced from -2 mm Hg to -20 to -40 mm Hg after injury, which will profoundly increase transcapillary filtration. Following the onset of fluid therapy, Pif increased to slightly positive values. In control, colloid osmotic pressure in plasma (COPp) was 20.6 +/- 0.4 mm Hg and in interstitial fluid (COPif) 13.7 +/- 0.3 mm Hg (means +/- SEM). The transcapillary oncotic pressure gradient (COPgrad = COPp-COPif) was 6.9 +/- 0.4 mm Hg. Following nonresuscitated thermal injury, COPp declined to 18-19 mm Hg (P less than 0.05) and COPif was reduced to 10.4 +/- 0.5 mm Hg (P less than 0.05). Fluid therapy by lactated Ringer's markedly reduced COPp (12.3 +/- 0.3 mm Hg; P less than 0.05), and COPgrad was almost abolished (2.6 +/- 0.7 mm Hg; P less than 0.05). In contrast, plasma infusion maintained COPp, whereas COPgrad increased significantly (11.1 +/- 1.2 mm Hg; P less than 0.05). Noncolloid saline solutions have been preferred for the initial fluid therapy for burns. The present study provides evidence that this will reduce both COPp and COPgrad, a situation in which edema formation will be favored.
Animals↗
Effect of longstanding venous stasis and hypoproteinaemia on lymph flow in the rat tail.
This study was performed to provide information on the determinants of lymph flow by comparing the effect of venous stasis and hypoproteinaemia in the rat tail. This low-compliant tissue was chosen in an attempt to induce preferential changes in interstitial pressure or volume. The removal rate (kAlb) of 125I-labelled human serum albumin (I-HSA) injected subcutaneously was monitored with external gamma-counting equipment and used as a measure of lymph flow. Interstitial fluid hydrostatic pressure (Pi) was measured with wick-in-needle technique, and interstitial fluid was collected post mortem by dry wicks. Colloid osmotic pressure of plasma (COPp) and wick fluid (COPi) was measured with a colloid osmometer. In a separate group of experiments, 51Cr-EDTA and [125I]HSA were used to measure the interstitial fluid volume. Venous stasis, induced by bilateral ligation of the external tail veins, increased interstitial fluid hydrostatic pressure from 1.7 to 16 mmHg and kAlb from 0.030 to 0.063 h-1, whereas tail circumference was nearly constant. Interstitial volume averaged 1.17 ml/g dry weight in control animals and 1.27 ml/g during increased venous pressure. Daily injections of aminonucleoside in salt-loaded rats (0.3% NaCl as drinking water) reduced colloid osmotic pressure of plasma from 19.1 to 8.5 mmHg and of wick fluid from 11.2 to 2.9 mmHg, while interstitial fluid hydrostatic pressure increased to 5.2 mmHg. The removal rate of 125I-labelled human serum albumin increased to 0.113 h-1, compared to 0.051 h-1 in salt-loaded controls. The interstitial volume showed a marked increase in salt-loaded hypoproteinaemic rats, 1.75 ml/g dry weight, compared to 1.30 ml/g in salt-loaded controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver.
The catabolism of hyaluronan has been studied by injecting hyaluronan, labelled with 125I-tyramine cellobiose (125I-TC), subcutaneously into the hindpaw of rabbits. Following endocytosis, 125I-TC remains in the cells at the site of uptake, allowing localization of the site of catabolism. At 6 h after subcutaneous injection, 65% of the injected radioactivity was recovered. The skin at the injection site contained 47%, the popliteal gland at the side of injection 10%, and the liver 8% of the injected dose. At 48 h the three organs contained 40% of the injected dose with 17% in the skin, 10% in the lymph node and 13% in the liver. The decline in recovery could be accounted for by urinary excretion of the tracer, implying that some tracer had been released from the cells after endocytosis. Chromatography revealed that over 85% of 125I-TC-hyaluronan in the lymph nodes and liver was of low molecular mass throughout the experiment. In skin, 4% of the injected tracer was recovered with low molecular mass at 6 h, increasing to 12% of injected dose at 24 and 48 h. Thus, a minimum of 12% of the injected tracer was catabolized per 24 h at the skin injection site. If cells in skin are responsible for the subsequent release of tracer, as seen from the decrease in recovery of the injected dose, another 10-15% of the tracer could have been catabolized locally in the skin per day. The major part of the hyaluronan injected in the skin was, however, catabolized by lymphatic removal and subsequent degradation in local lymph nodes and liver.
Animals↗
Increased negativity of interstitial fluid pressure during the onset stage of inflammatory edema in rat skin.
Interstitial fluid pressure (Pif) was measured in skin of pentobarbital-anesthetized rats during anaphylaxis toward dextran and after subdermal injection of histamine by using sharpened glass capillaries (tip diam 5-7 microns) connected to a servo-controlled counterpressure system. Control Pif averaged -1.5 mmHg (SD = 1.0). After intravenous dextran Pif in the rat paw fell transiently to -3 mmHg up to 20 min and thereafter increased to +1-2 mmHg when edema had developed. To study the full magnitude of the increased negativity of Pif, circulatory arrest was induced 1 min after dextran injection. This procedure prevents accumulation of edema that will cause underestimation of Pif. In this group Pif fell to about -10 mmHg in 20 min and remained at this level throughout the observation period of 90 min. Subdermal injection of 1-10 micrograms histamine in 10 microliters saline reduced Pif to about -6 mmHg within 5 min after injection. Injection of 10 microliters saline increased Pif by +2 mmHg. Indomethacin or cyproheptadine did not alter the response in the above situations. The increased negativity in Pif of about 6-8 mmHg will add directly to normal transcapillary net filtration pressure of 0.5 mmHg and increase the latter pressure 10-20 times.
Anaphylaxis↗
Analysis of lymphatic protein flux data. V. Unique PS products and sigma dS at low lymph flows.
The selectivity of the capillary membrane to protein (osmotic reflection coefficient, sigma d) can be measured at high transcapillary volume flow when the capillary membrane can be considered as a true sieve. However, the diffusive capacity of the membrane (permeability-surface area product, PS) for macromolecules has not been directly measured, only estimated by assuming that transcapillary volume flow was zero. Based on unique properties of the Peclet number, a parameter that describes the ratio of solute convective flux relative to diffusive capacity, we have developed three new techniques using lymph protein fluxes to estimate a unique PS product that is independent of transcapillary fluid flux. Two of these techniques require a measure of sigma d when the ratio of protein concentration in lymph relative to plasma is equal to (1- sigma d), which occurs at high capillary filtration rates. However, the third method allows both sigma d and the PS product to be determined at relatively low lymph flow rates, eliminating the need for high capillary pressures to determine sigma d. For each protein, these techniques yield an estimate of PS and sigma d for the total membrane. However, by analysis of several different sized proteins and estimation of small- and large-pore volume flows, sigma d and PS can be determined separately for the small- and large-pore pathways. These techniques for estimating sigma d and PS were evaluated by modeling the total solute flux of albumin and immunoglobulins G and M in a heteroporous membrane.
Animals↗
Albumin transport across pulmonary capillary-interstitial barrier in anesthetized dogs.
To evaluate albumin transport across the pulmonary capillary endothelial and interstitial barriers, we simultaneously measured blood-to-tissue (QA,t) and blood-to-lymph (QA,l) clearances of 125I-radiolabeled albumin as well as endogenous albumin clearance (Qa,l) in the canine lung in vivo (n = 10). Steady-state prenodal lung lymph flows (Qw,l) and protein clearances were measured over a 2-h period at a constant capillary pressure (Pc, 13-33 cmH2O). Comparison between QA,t and QA,l as a function of Pc suggests that little of the albumin that crossed the capillary wall remained in the lung tissue, with most leaving in the lymph. Qw,l increased significantly as Pc increased, but lung tissue water was minimally affected. From the ratio of the clearance-Pc slopes for albumin and water, the albumin reflection coefficient was estimated to be 0.81 using QA,l and Qw,l and 0.56 using Qa,l and Qw,l. The permeability surface area product for the sum of blood-to-tissue and blood-to-lymph fluxes of labeled albumin (QA,t + QA,l) was 31 +/- 9 microliters/min, whereas that calculated from the blood-to-lymph flux of endogenous albumin (Qa,l) was 97 +/- 22 microliters/min. These data suggest that 1) both tissue and lymph accumulations of albumin must be considered when microvascular permeability is evaluated using protein tracers; 2) lymph clearance, but not tissue accumulation of albumin, was filtration dependent; and 3) lymph flow was an important contributor to the safety factor against edema formation over a moderate range of capillary pressures.
Albumins↗
Permeability-surface area product and reflection coefficient of the parietal pleura in dogs.
The parameters describing the permeability of the parietal pleura to liquid and total plasma proteins were measured in five anesthetized adult dogs. Small areas of parietal pleura (approximately 1 cm2) and the underlying endothoracic fascia were exposed through resection of the skin and the intercostal muscles. The portion of the thorax containing the pleural windows was removed from the chest and fixed over a bath of whole autologous plasma, the inner parietal pleural surface facing the bath. Small hemispheric Perspex capsules (surface area 0.28 cm2) connected to a pressure manometer were glued to the pleural windows; a subatmospheric pressure was set into the capsule chamber to create step hydraulic transpleural pressure gradients (delta P) ranging from 5 to 60 cmH2O. Transpleural liquid flows (Jv) and protein concentration of the capsular filtrate (Cfilt) and of the plasma bath were measured at each delta P. The transpleural protein flux (Js) at each delta P was calculated by multiplying Jv by the corresponding Cfilt. The hydraulic conductivity (Lp) of the parietal pleura was obtained from the slope of the Jv vs. delta P linear regression. The average Lp from 14 capsules was 9.06 +/- 4.06 (SD) microliters.h-1.cmH2O-1.cm-2. The mathematical treatment of the Js vs. Jv relationship allowed calculation of the unique Peclet number at the maximal diffusional protein flux and a corresponding osmotic permeability coefficient for plasma protein of 1 x 10(-5) +/- 0.97 x 10(-5) cm/s. The reflection coefficient calculated from the slope of the linear phase of the Js vs. Jv relationship was 0.11 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Elevated hyaluronan blood concentrations in severely burned patients.
Hyaluronan (HYA) is a polysaccharide found in the interstitial matrix in most tissues. HYA is removed by lymphatic drainage and degraded in local lymph nodes and in the liver. Its normal concentration in human plasma is very low (10-100 micrograms l-1). Following major burn injury in sheep plasma HYA can increase to levels tenfold greater than normal. In the present study, serum HYA concentrations were determined in 10 severely burned patients (burn size: 28 +/- 5% of body surface area (mean +/- SEM)). Serum HYA concentration was 206 +/- 71 micrograms l-1 at 24 h post-injury, twice the upper normal HYA concentration, and remained moderately elevated for the first week post-injury. It appears that mobilization and transport of HYA from the tissues is increased after thermal injury. Increased systemic concentrations of an interstitial matrix component have not previously been reported in burn patients. Possibly, plasma concentrations of connective tissue components might serve as indicators of the severity and extent of burn injuries.
Adult↗
Microvascular exchange during burn injury: IV. Fluid resuscitation model.
The present work is a continuation of studies concerned with mathematical modelling and simulation of microvascular fluid and protein exchange following burn injuries [Bert et al.: Circulatory Shock 28: 199-219, 1989: Bowen et al.: Circulatory Shock 28: 221-233, 1989]. The model has been extended to include the effects of different types of fluid resuscitation on the circulatory and microvascular exchange systems. The model and a statistical fitting procedure were used to find the ranges of fitting parameter values that best describe the changes in interstitial fluid volume and protein mass as well as transcapillary protein extravasation for three sets of experiments (no resuscitation, resuscitation with Ringer's or resuscitation with plasma). Typical changes in mass exchange related parameters postburn that resulted in simulation predictions which were a good fit to the experimental data include: an increase in the large pore pathway for protein of 100 times in the injured skin and 5 times in non-injured skin and skeletal muscle, an increase in fluid filtration coefficients in injured skin of 10 times and an instantaneous decrease of 50% in the area available for exchange in injured skin at the time of the burn.
Burns↗
Chromogranin A: osmotically active fragments and their susceptibility to proteolysis during lysis of the bovine chromaffin granules.
Osmotically active fragments of chromogranin A (Chr A) were studied in lysates from bovine chromaffin granules (CG) disrupted in the presence or absence of inhibitors of endogenous proteolytic activities. The effects of various methods of lysis were examined by micro-osmometry, PAGE-SDS electrophoretic techniques and immunoblots with polyclonal anti-Chr A sera. Osmotically active 'small' Chr A fragments (below 30 kDa) were conspicuous in lysates containing cocktails of leupeptin, pepstatin A, pHMB, PMSF and aprotinin. The osmotically inactive native Chr A in the 68-100 kDa range and the osmotically active fragments below 47 kDa were degraded in lysates at neutral or acid pH in the absence of inhibitors. However, degradation of the native Chr A and intermediates below 47 kDa could be prevented by extraction directly from intact CG, notably in cold or boiling distilled water. On the other hand, the main product after large-scale extraction of CG in 1 M acetic acid (pH 1.9, 100 degrees C) was a novel, osmotically active fragment (22 kDa), immunostaining only for the N-terminal sequence (Chr A1-40). The heat-stable fraction (Mr,n 23 kDa) exhibited concentration-independent colloid osmotic pressures even in the absence of phosphate, a property which may distinguish this N-terminal-containing fragment from the larger intermediates, probably containing the pancreastatin sequence, and other regions at the C-terminal side of the prohormone molecule. The functional roles of these osmotically active intermediates in the processing of Chr A are not yet known.
Acids↗