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Am J Physiol Heart Circ Physiol 290: H2108-H2115, 2006. First published December 22, 2005; doi:10.1152/ajpheart.00395.2005
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Changes in plasma protein extravasation in rat skin during inflammatory challenges evaluated by microdialysis

B. Å. Borge, V. V. Iversen, and R. K. Reed

Department of Biomedicine, Section of Physiology, University of Bergen, Bergen, Norway

Submitted 21 April 2005 ; accepted in final form 15 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Docetaxel and prostaglandin E1 (PGE1) increase transcapillary albumin extravasation and reduce interstitial fluid pressure in the skin. In this study the microdialysate concentration (Cm) of 125I-labeled human serum albumin (125I-HSA) and different-sized endogenous plasma proteins (EPP) was compared to evaluate changes in transcapillary extravasation of plasma proteins. 125I-HSA was also used to estimate changes in the specific activity of albumin. Extravasation of 125I-HSA and EPP from plasma to interstitium in the rat skin was compared during continuous administration of docetaxel and PGE1 by using microdialysis in anesthetized rats. Also, 20 ml of Ringer solution (RS) were injected intravenously during 10 min in a separate group. Two hollow plasmapheresis fibers (3 cm, cut off 3,000 kDa), one acting as control, were placed subcutaneously on the back skin and perfused with RS (5 µl/min, 140 min, collected every 10 min). The size of the different EPP was estimated to be 73, 65, 56, 47, and 39 Å, separated by a size-exclusion high-performance liquid chromatography column and quantified by UV detection (280 nm). Docetaxel (0.5 mg/ml, n = 5) increased Cm of 125I-HSA and EPP of sizes 73, 65, 56, and 39 Å significantly (P < 0.05) compared with control. PGE1 (20 µg/ml, n = 6) increased Cm of 125I-HSA significantly (P < 0.05) but none of the different-sized EPP was increased compared with control. Intravenous RS (20 ml, n = 6) increased Cm of 125I-HSA and increased all the different-sized EPP significantly (P < 0.05) compared with control. Although the microdialysis method is able to monitor qualitative changes in capillary permeability, a quantitative determination of the capillary reflection coefficient or permeability-surface area product was not possible, because steady state between plasma and dialysate was not achieved during the measurement period. The different pattern of extravasation of EPP and 125I-HSA after docetaxel, PGE1, and RS indicates increased interstitial transport rate and/or increased capillary permeability after docetaxel and RS, whereas PGE1 seems to increase transcapillary fluid flux without altering the permeability.

high-performance liquid chromatography; prostaglandin E1; docetaxel; transcapillary transport

QUANTIFICATION of macromolecular passage from plasma to interstitial fluid may give important information of the integrity of the capillary barrier. Many pharmacological agents and pathological conditions have been shown to alter the permeability and/or protein flux across the capillaries in subcutaneous tissue (39)

Docetaxel is a microtubule fixating agent and has previously been demonstrated to lower interstitial fluid pressure (Pif) and increase the extravasation of 125I-labeled human serum albumin (125I-HSA) (5, 17). The same effects have been observed after administration of PGE1 (3, 17), in addition to its effects as a vasodilator (23). Reduction of Pif and increased vasodilatation are both properties that contribute to increase transcapillary fluid flux (Jv) (2, 18). Prostaglandins have also been shown to increase Jv without altering the ratio of albumin in lymph versus plasma (19), and the increased protein flux produced by these compounds and other vasodilators was believed to result from an increased exchange-vessel surface area and augmentation of fluid filtration. Inflammatory agents like histamine and bradykinin reduce the selectivity of the capillary barrier in addition to increasing transcapillary fluid flux (6, 36, 43), and these changes were attributed to an alteration of the pathway for large molecular transport. Docetaxel also appears to increase transcapillary protein transport (17, 37). One of the parameters important in this context is the capillary reflection coefficient for proteins ({sigma}).

Interstitial fluid is not directly accessible in normally hydrated tissues, and several methods have been developed for isolation of such fluid, like lymph sampling, centrifugation method (41), microdialysis, and the wick method.

Previous attempts to measure changes in capillary permeability in subcutaneous microcirculation have mostly been performed by either measuring lymph-to-plasma ratio of plasma proteins after increased venous pressure (11, 12, 31, 39) or measuring tissue accumulation of tracer macromolecules (29, 30). Compared with studies of lymph flow and lymph protein concentration for determination of capillary permeability, the microdialysis method has the advantage that the sampling site is much closer to the capillary wall. This should markedly reduce the time required to obtain a steady state and allow better time resolution of the acute changes in the capillary wall. In addition, the method allows continuous measurement of the response with simultaneous administration of the treatment. Also, the method has the advantage that the whole time course can be obtained for the event at the capillary-to-tissue exchange and not only a single point, which is the case when measuring blood-to-tissue exchange with radioactive tracers.

The aim of this study was to evaluate the use of skin microdialysis to estimate changes in capillary selectivity in rat back skin during different inflammatory challenges. This was performed by continuous measurement of changes in the relative concentration of different-sized endogenous plasma proteins (EPP) and 125I-HSA in the dialysate. The different pattern of extravasation between different-sized EPP and 125I-HSA was compared and utilized for evaluation of changes in the transport of plasma proteins from plasma to the microdialysis probe. In addition, 125I-HSA was used to measure specific activity of albumin in dialysate and plasma. Overhydration experiments with injection of 20 ml Ringer solution (RS) were also performed to evaluate the effect of increased convective fluid flux on interstitial concentration of different-sized EPP when the capillaries and interstitium were unaffected by inflammatory agents.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Theory. Transport of macromolecules across the capillary can be described by the Patlak equation, which describes solute transport by simultaneous convection and diffusion in the same pathway (28):

Formula 1(1)
Here Js and Jv are transcapillary protein flux and fluid flux, respectively, Cp and Ci are protein concentrations in plasma and interstitial fluid, respectively, and PS is the permeability-surface area product. x is the so-called Peclet number and describes the convective flux relative to the diffusive capacity of the membrane:

Formula 2(2)
Under normal physiological conditions, protein transport across the capillary is influenced by both convection and diffusion. By increasing Jv, protein transport by diffusion becomes negligible (35) because the term modifying the PS product [x/(ex – 1)] decreases with increasing Jv. This causes the concentration of plasma proteins in capillary filtrate (Cf) to approach a constant filtration rate independent value described by Eq. 3 (4, 39).

Formula 3(3)
The following four criteria needed to be fulfilled for our experimental model to generate data suitable for estimation of changes in {sigma}. 1) Plasma concentrations of the different EPP studied were unaffected by the local administration of inflammatory agents through the microdialysis probe. 2) The concentrations of different EPP in the dialysate were proportional to the true interstitial concentration, and the relative recovery of the probe toward interstitial EPP was constant throughout the measurement. Because we measured changes in relative values, it was not necessary for recovery to be the same between every protein species. 3) A steady state is achieved between the concentration of plasma proteins in the transcapillary fluid, interstitial fluid, and the dialysate during the measurement period. 4) After administration of docetaxel, PGE1, or 20 ml RS, Jv increases from its normal value until Cf/Cp approaches a constant value (filtration independent). The contribution of diffusion to Js is then expected to be negligible (see Eq. 3).

If the above criteria are fulfilled, then {sigma} could be estimated from

Formula 4(4)
Because Cp under these circumstances is constant and the relative concentration of plasma proteins in the dialysate (Cm) is proportional to Ci, changes in Cm are directly proportional to changes in (1 – {sigma}). An increase in the relative concentration of different-sized EPP could therefore be interpreted as a reduction of {sigma} for proteins of this size.

Animals. Female Wistar rats were obtained from Taconic M&B (Denmark) and Harlan (Oxon, UK). They were fed a standard diet and tap water ad libitum. The rats weighed 200–250 g and were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), with supplemental doses when required. Rectal temperature was measured and maintained at 37.0 ± 1°C by a heating pad during the experiment. The experiments in this study have been performed with the approval of, and in accordance with, the regulations laid down by the National Animal Research Authority.

Microdialysis. Microdialysis was performed as described by Schmelz et al. (36) and modified by Iversen et al. (17). Two hollow plasmapheresis fibers with 3 cm membrane (cut off 3,000 kDa) were placed subcutaneously 4 cm apart on the back of anesthetized rats. After the surgical procedures were completed, each fiber was perfused with RS (Fresenius, Germany) at a flow rate of 5 µl/min by a 2-ml glass syringe in a CMA/100 microdialysis pump (CMA, Stockholm, Sweden) for 60 min to stabilize. After 50 min of stabilization, the animals received an intravenous injection containing 0.2 ml 125I-HSA (0.4 MBq), which circulated for the remaining 10 min of the initial stabilizing period. Thereafter, the perfusate was sampled for 40 min to establish a baseline followed by an experimental period of 100 min. The perfusate was collected in intervals of 10 min into 0.8 ml high-performance liquid chromatography (HPLC) vials (Gilson, Mildred, WI). The RS was vacuum degassed before use to avoid accumulation of air bubbles in the perfusion fluid, which could create a flow hindrance. The sampling vials were analyzed for radioactivity in a gamma counter (LKB Wallac, Turku, Finland) after the experiment and then frozen (–20°C) for later analysis by HPLC.

HPLC of dialysate. The concentrations of different-sized EPP in the dialysate were determined by HPLC analysis. EPP in the perfusate were separated by a TSK-Gel Super SW3000 size-exclusion HPLC column (TOSOH Bioscience; Stuttgart, Germany) and quantified by UV detection at 280 nm (Spectraseries UV10; San Jose, CA). The UV signal was digitized, sampled at 2 Hz, and analyzed on a computer by using ChromQuest (version 2.51, Thermoquest; San Jose, CA). HPLC buffer (50 µl) (in M: 0.1 Na2SO4, 0.72 NaH2PO4, and 0.28 K2HPO4; pH 6.7) was added to the sample vials before the sample was injected on the HPLC system (20 µl loop, run time 20 min, perfusion rate 0.35 µl/min) by using a Gilson 234 autosampler (Gilson). The different-sized EPP were grouped into five main peaks that were estimated to consist of proteins with a Stoke-Einstein radius of approximately 73, 65, 56, 47, and 39 Å (Fig. 1) based on measurement of the column eluent volume for proteins with known diameter, using a gel filtration calibration kit (Pharmacia Biotech; Piscataway, NJ). The average amount (measured by area under the curve) of 125I-HSA or EPP in the four first vials (0–40 min) of each experiment was defined as 100%, and all subsequent values were calculated as percentage of this average (Cm). In addition, absolute values of albumin concentration (corresponding to the peak at 39 Å) were calculated using external albumin standards (Sigma-Aldrich; St. Louis, MO). Calculations of absolute values of the other peaks were not deemed possible because each peak was expected to be a mixture of many different proteins with approximately the same size. The change in plasma concentration of 125I-HSA and different-sized EPP was measured after administration of docetaxel and intravenous injection of RS. The amount of 125I-HSA or EPP in the first blood samples (collected at 30 or 20 min, respectively) was defined as 100%, and the values in all subsequent blood samples were expressed as a percentage of this value (CP). The specific activity was estimated as the amount of radioactivity (counts per minute) divided by the amount of native albumin (mg/ml) in a sample. In a closed system with no metabolism, the specific activity becomes equal in all compartments at tracer steady state. The plasma-interstitial system, however, is an open system because albumin is catabolized in the plasma compartment. Thus, if steady state was achieved during our experimental protocol, the ratio of specific activity between dialysate and plasma was expected to be above 1.


Figure 1
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  		Fig. 1. Chromatogram for a sample collected from control probe after 70 min and eluated (Ve) through the TSK-Gel Super SW3000 size-exclusion column. Peak: 1) 73 Å, 2) 65 Å, 3) 56 Å, 4) 47 Å, and 5) 39 Å (albumin).

 
The percentage difference in albumin between the two probes (DABP) in the same animal was calculated to express the variability between the two probes under identical conditions. DABP was calculated for every sample of dialysate collected at 0–40 min (baseline). All the animals were treated in the same manner during the baseline measurement. The largest of the two sample values was divided by the smaller, and the average value of the four measurements in each animal was used to calculate means ± SE.

Formula 5(5)

Experimental protocol for docetaxel and PGE1. Local administration of docetaxel or PGE1 through the microdialysis probe was used to induce a local and acute inflammation reaction in the area around the probe. Cannulation of the left femoral vein was performed for intravenous injections of 125I-HSA (Institute of Energy Technique, Kjeller, Norway). Cannulation of the right carotid artery was also performed in animals treated with docetaxel for sampling of arterial blood (100–200 µl per sample) at 30, 70, 110, and 140 min. After the baseline period (0–40 min), one of the fibers was swapped by a liquid switch (CMA/110) from a syringe containing RS to a syringe containing docetaxel (0.5 mg/ml, n = 5) or PGE1 (20 µg/ml, n = 6). Administration of docetaxel or PGE1 through one of the microdialysis fibers continued during the rest of the experiment (40–140 min), whereas the other fiber served as control and was perfused by RS.

Experimental protocol for injection of 20 ml RS. Intravenous injection of RS was performed to create a transient increase in Jv as a result of overhydration. Cannulation of the left femoral vein was performed for intravenous injections of 125I-HSA and 20 ml RS and cannulation of the right carotid artery for sampling of arterial blood (100 µl per sample) at 20, 50, 60, 90, 120, and 140 min. Because injection of 20 ml RS will increase diuresis, the kidneys were ligated to avoid fluid loss from plasma to urine during the experiment. RS was continuously perfused through both microdialysis fibers (5 µl/min) during the entire experiment.

After the baseline period (0–40 min), 20 ml of RS were injected intravenously during a time period of 10 min (n = 6). The relative concentration of 125I-HSA and different-sized EPP in the dialysate were divided by the relative changes in plasma for the same protein after injection of 20 ml RS.

Formula 6(6)
This correction was performed because injection of 20 ml RS resulted in a large increase in plasma volume and thereby a significant reduction in concentration of all plasma proteins (Fig. 2B). Unless this factor was taken into account, the measurement of relative changes of EPP in the dialysate after injection would be underestimated. Perfusate collected at 40–50 min was corrected with plasma collected at 50 min; perfusate 50–70 min/plasma 60 min; perfusate 70–110 min/plasma 90 min; perfusate 110–130 min/plasma 120 min; perfusate 130–140 min/plasma 140 min.


Figure 2
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  		Fig. 2. Effect of local administration of docetaxel (n = 5, A) and injection of 20 ml Ringer solution (n = 6, B) on the relative concentration changes in plasma of different-sized endogenous plasma proteins and 125I-labeled human serum albumin (125I-HSA) compared with plasma collected at 30 or 20 min, respectively (baseline, 100%). Values are means ± SE. *Significantly different (P < 0.05) from samples collected at 30 min. For B, all values in samples collected after 20 min were significantly less (P < 0.05) than samples collected at 20 min.

 
Statistical analysis. Data are given as means ± SE. The measured values in the last four microdialysis samples (110–140 min) were averaged before the results were analyzed. A normality and equal variance test were performed, and the datasets that passed both tests (P < 0.05) were further analyzed with one-way ANOVA followed by post hoc Bonferroni test to correct for multiple comparisons between the different groups (see results in Fig. 3, B–F, and Fig. 5). Data that failed to pass the tests were analyzed with Kruskal-Wallis one-way ANOVA on ranks followed by post hoc Dunn's test to correct for multiple comparisons between the different groups (see results in Fig. 3A). One-way ANOVA followed by post hoc Bonferroni to correct for multiple comparisons were also performed within each experimental group, between the different-sized EPP, illustrated in Fig. 4. The relative changes in plasma protein were analyzed with one-way repeated ANOVA followed by post hoc Bonferroni for comparisons to samples collected at 20 or 30 min. Differences were accepted as statistically significant when P < 0.05. The statistical analysis was done using a statistical package (SigmaStat 2.03, Systat software).


Figure 3
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  		Fig. 3. Effect of local administration of docetaxel (40–140 min, n = 5) or PGE1 (40–140 min, n = 6) or intravenous injection of 20 ml Ringer solution (40–50 min, n = 6) or local perfusion of Ringer solution (control, n = 11) on the extravasation of 125I-HSA (A) and relative concentration changes in dialysate of endogenous plasma proteins (Cm) of size 73 Å (B), 65 Å (C), 56 Å (D), 47 Å (E) or 39 Å (F) compared with baseline (0–40 min, 100%). Values are means ± SE. Each experimental group was considered significantly different when the average of the 5 last samples (100–140 min) achieved P < 0.05: *compared with control; §compared with PGE1; #compared with infusion of 20 ml Ringer solution.

 

Figure 5
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  		Fig. 5. Effect of local administration of docetaxel (n = 5) or intravenous injection of 20 ml Ringer solution (n = 6) on the ratio of specific activity of albumin between dialysate and plasma. *Each experimental group was considered significantly different from control if the average of the 5 last samples (100–140 min) achieved P < 0.05.

 

Figure 4
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  		Fig. 4. Effect of local administration of docetaxel (n = 5) or PGE1 (n = 6) or intravenous injection of 20 ml Ringer solution (n = 6) on relative concentration changes of different-sized endogenous plasma proteins in the dialysate compared with control as a function of size in the range of 39 of 73 Å. Values are means of the 5 last sampled values (100–140 min) ± SE. P < 0.05: *compared with 39 Å; §compared with 47 Å. Numbers indicate mean values.

 

   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Changes in plasma. Albumin concentration in plasma averaged 49 ± 2 mg/ml in samples collected at 20 and 30 min (n = 11, means ± SE). Local administration of docetaxel significantly reduced the relative concentration of EPP of size 73, 65, 56, and 47 Å in blood samples collected after 110 min compared with the samples collected at 30 min to ~92% (P < 0.05, Fig. 2A). Injection of 20 ml RS at 40 min significantly reduced the concentration of all EPP to about 60–80% in samples collected after 50 min (P < 0.05, Fig. 2B). After both local administration of docetaxel and intravenous injection of 20 ml RS, the concentration of 125I-HSA decreased significantly (P < 0.05) throughout the measurement period compared with samples collected at 20 and 30 min (basal period).

Albumin concentration. Total albumin concentration and DABP in the dialysate were measured during the basal period (0–40 min, Table. 1). The mean values of albumin were in the range of 0.48 ± 0.06 to 1.19 ± 0.27 mg/ml. The mean values of DABP averaged from about 30% to 57%. There were no significant changes between the different experimental groups. The absolute concentration of albumin in the dialysate was around 1 mg/ml (Table 1) and consequently 5–10% of what should be expected in interstitial fluid based on measurements using the wick method or lymph sampling. Also, as seen from the variation in albumin concentration between probes, it would be incorrect to use a standard value to correct the measured value for a low recovery and high interfiber variability. Therefore, experimental values (40–140 min) were compared with their own baseline values (0–40 min) obtained with the same probe.


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  		Table 1. Albumin concentration in the dialysate collected during baseline period for different experimental groups

 
Control groups. All fibers perfused with only RS during the entire measurement period (0–140 min) were treated as one large control group (n = 11; 5 controls from the PGE1 experiments and 6 controls from docetaxel experiments). Measurement of the perfusate from control fibers revealed a steady increase in the concentration of 125I-HSA throughout the measurement period (Fig. 3A). The concentration of all the EPP showed a slight and gradual reduction with time throughout the entire experiment (Fig. 3, B–F).

Experimental groups. The extravasation of 125I-HSA increased significantly (P < 0.05) compared with control after local administration of docetaxel and PGE1 and after intravenous injection of RS (Fig. 3A). The large SE observed in the docetaxel group was caused mainly by results from one "outlier" animal (animal 2). Local administration of docetaxel through the microdialysis probe significantly increased (P < 0.05) the microdialysis concentration of EPP of size 73, 65, 56, and 39 Å by 40 to 180% above control (Fig. 3, B–F). The degree of increase was correlated to the size of the EPP in the order of 65 Å > 73 Å > 56 Å > 39 Å > 47 Å (Fig. 4). In contrast, intravenous injection of 20 ml RS increased all the different-sized EPP to an approximately constant level (45–65%). The same pattern was observed after local administration of PGE1 (increased nonsignificantly to 10–30% above control). In both the docetaxel and RS experiments, the increase was rapid and stabilized 20–30 min after injection.

Specific activities of albumin. None of the experimental groups achieved values close to the steady-state value of 1.

The ratio of specific activities after both administration of docetaxel and injection of RS increased significantly compared with control and reached a maximum at ~0.5–0.6 (Fig. 5) after 140 min. The control group reached a maximum of ~0.2.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The overall aim of the present study was to use microdialysis to evaluate changes in capillary permeability. To achieve this, we used inflammatory mediators as a tool to perturb the normal capillary permeability. Administration of docetaxel significantly increased the extravasation of 125I-HSA and the concentration of different-sized EPP in the dialysate. The magnitude of increase for each EPP was greater for the larger protein components of plasma (73, 65, and 56 Å) than for albumin. The increase was such that the permeability of the capillary wall and/or tissue properties in the interstitium must have been altered to explain our observations. Similar results have previously been reported for other inflammatory agents, like histamine (6), and was explained by increased capillary transport of large proteins. Treatment of cancer patients with docetaxel have also been shown to increase capillary protein extravasation from plasma to skin (37). The result could also be explained by an increased PS area due to vasodilatation or recruitment of capillaries in the vicinity of the probe and thereby increased transcapillary diffusion of EPP. However, it was not resolved whether the sustained elevation of different-sized EPP and 125I-HSA was due to an increase in protein transport alone or if an increase in interstitial volume fraction also contributed. If skin edema occurred during the experiment, the transport rate of plasma proteins through the interstitium and into the probe could increase (13).

Docetaxel is a microtubule fixating agent and has been demonstrated to lower interstitial fluid pressure (Pif) and increase the extravasation of 125I-HSA (5, 17). The same effects have been observed after administration of PGE1 (3, 17) in addition to its effects as a vasodilator (23). However, the mechanism responsible for the observed changes in protein extravasation from plasma to dialysate seems to differ between docetaxel and PGE1. Reduction of Pif and vasodilatation are properties that both were expected to increase Jv. The increase in 125I-HSA extravasation after administration of PGE1 without any increase in EPP could be the result of an increase in Jv without any changes of the capillary permeability toward proteins. This effect of PGE1 would be in accordance with previous reports, where PGE1 increased transcapillary fluid transport without affecting the capillary selectivity or the PS area (19). Our method was not regarded suitable for quantitative estimation of either the PS area product or Jv. Previous studies on dogs have shown an increased lymph flow but no alteration of either the PS area or lymph-to-plasma concentration ratio for total plasma proteins after subcutaneous injection of PGE1 (19). Inflammatory agents like histamine, on the other hand, have been reported to increase lymph flow, lymph-to-plasma concentrations ratio, and the PS area for total proteins from 2 to 10 times compared with control (19, 20). There is controversy concerning the amount of increase in Jv necessary to achieve a dominantly convective protein transport with negligible diffusive transport. Studies in dog paw (31) and rabbit skin (40) indicate that an increase of two to four times above normal should be adequate. The use of local administration through the microdialysis probe and subsequent HPLC analysis of the dialysate did not allow determination of changes in Jv. This is a a major disadvantage compared with previous studies using lymph. We were therefore unable to determine whether criteria 4 were achieved, which made it difficult to conclude if the observed change in protein extravasation was due to changes in {sigma}.

Effect after injection of 20 ml RS. If Jv increases while {sigma} remains constant, the concentration of EPP in the dialysate was expected to drop because the contribution of diffusion to transcapillary protein transport is reduced with increasing Jv (2, 21, 32, 39). Injection of 20 ml RS increased the extravasation of 125I-HSA as expected due to an increase in Jv but also increased the concentration of all the different EPP in the dialysate. The increase was approximately the same for all the five EPP. The result does not seem to support an assumption of filtration rate independence and was unexpected because previous studies have shown a reduction in Ci/Cp of EPP after saline volume loading (25). There are several possible explanations for this aberration. First, intravenous injection of 20 ml RS was not expected to reduce {sigma}. It is more likely that an injection of large volume of fluids causes edema in the skin and thereby enhanced the transport of plasma proteins through the interstitium and into the probe (13) as previously discussed for docetaxel. Second, the insertion trauma after placement of the microdialysis probes could disturb the integrity of the capillary filtration barrier or the interstitium even after 60 min of stabilization (14, 15). This is supported by the slow decline of EPP in the control probes throughout the experimental period. However, Fadnes and coworkers (10) reported contamination of wick fluid from plasma proteins to be only 2–11% that of serum albumin measured by intravenous injection of 131I-HSA 30 min after wick insertion. The colloid osmotic pressure in wick fluid has also been shown to remain stable between 30 and 60 min after implantation of wick (42). It is likely that the insertion trauma and contamination from plasma proteins will be in the same range after insertion of a microdialysis fiber and will therefore not explain the aberration.

The slow decline of EPP in the control probe could also be the result of decreasing recovery over time. According to mathematical modeling, the initial steep extracellular concentration profile confined to a region in the vicinity of the probe was expected to reduce toward a more gradual shape extending into the tissue as analyte was removed (9). This effect would be caused by the constant perfusion through the probe (7, 36). Decreasing recovery over time during microdialysis would be reflected in a decreasing concentration of EPP in dialysate, and this behavior was observed in the control probes for all the different EPP.

The total volume of saline (~8–10% of body wt) was delivered with an infusion rate of 2 ml/min for 10 min. This distinguishes us from previous studies performed in rats, using continuous infusion rates of 0.4–0.66 ml/min (32) or 2 ml/min for 5 min followed by an continuous infusion at 0.4 ml/min (33) to increase Jv. The relatively large volume of 20 ml administrated over a short period of time may significantly increase venous pressure and cause venous congestion. An increase in venous pressure could cause a physical distortion of the subcutaneous capillaries and disturbed the integrity of the filtration barrier or forceful opening of ordinarily closed "large pores" in the microvascular membrane (34). This possibility is supported by earlier observations of increased plasma protein permeability at increased venous pressures (31).

Because steady state of EPP and 125I-HSA between plasma and interstitium was not achieved (Fig. 5 and discussion below), the adjusted value for Cm (Eq. 6) would be slightly overestimated. This effect combined with an alteration in the recovery of the probe, following structural changes in the extracellular matrix due to overhydration, was regarded the most likely explanation for the rapid increase in EPP in the dialysate after infusion of 20 ml RS. There is a controversy concerning the time required to reach a near steady state for fluid and proteins, between plasma and interstitium after overhydration with saline solution. Estimates based on previous experimental data ranges from 0.5–1 h (16, 24) to 3.5–5.5 h (25, 26).

Steady-state assessment. Determination of steady-state condition was important for the interpretation of the result. In a closed system with no metabolism, the specific activity becomes equal in all compartments at tracer steady state (27). The plasma-interstitial system is an open system because plasma proteins are catabolized by removal from the plasma compartment. Consequently, specific activity in the interstitium at steady state will exceed 1 due to the time delay required for plasma proteins to achieve steady state across the capillary barrier. The ratio of specific activity of albumin between the dialysate and plasma was significantly less than 1 in all cases, and hence steady state was not achieved. The specific activity for EPP of size 73, 65, 56, and 47 Å was not measured, but based on the results from albumin, we did not anticipate steady state for any of the remaining EPP. In comparison, previous studies measuring lymph concentration of plasma proteins and different-sized dextran achieved stabilization of lymph flow and protein/dextran concentration after 1.5–6 h of measurement (11, 25, 31, 40). Free iodide should not be a source of error in the measurements because the albumin tracer contained <3% free iodide. On injection into the circulation, the majority of the iodide remains in the circulation because it is attached to the albumin. However, the free iodide is distributed in a space larger than extravascular space and consequently is <0.3% in plasma very shortly and thereafter remains a constant background throughout the experiment because of low renal clearance. Protein flux from plasma to interstitium and further into the microdialysis probe was not expected to increase the ratio of free iodide.

Before the experiment starts, EPP in plasma and interstitium was in steady state. The level of endogenous albumin (39 Å) in the interstitium of normal skin is approximately 1/2 to 2/3 of the level in plasma (40–50 mg/ml) (1). This level gradually changes during the measurement period as a result of experimentally induced changes in {sigma}, Jv, and diffusion across the capillaries. On the other hand, injection of 125I-HSA was performed only 10 min before the measurement started. As the measurement starts, the level of 125I-HSA in the interstitium is very low. The result is the gradual increase in 125I-HSA observed throughout the experiment in the control group in Fig. 3A, and is the reason for the different pattern of extravasation between endogenous albumin and 125I-HSA. The interstitial concentration of 125I-HSA was determined exclusively by changes in total transport of albumin across the capillary. Differences in extravasation of 125I-HSA and EPP after docetaxel, PGE1, and 20 ml RS allowed inferences about the mechanism responsible for the changes to be made.

Fulfillment of other criteria assumed in our model. The requirement of constant Cp for different-sized EPP was assumed to be fulfilled during perfusion of docetaxel and PGE1 through the microdialysis probe. The gradual decrease in EPP concentration in plasma after local administration of docetaxel (Fig. 2A) was less than 10% after 110 min and was not expected to result from a systemic effect of the small amount of docetaxel perfused through the microdialysis probe. The reduction had little impact on our measurements and was more likely explained by the four times removal of 100–200 µl blood samples followed by dilution of blood as a 0.9% NaCl solution was injected to replace the collected volume. Alternatively, it could be a result of the anesthesia because it previously has been shown that pentobarbital sodium could increase the leakage of albumin from plasma (38). The albumin concentration in plasma at 20 and 30 min was fairly stable with small variation between individuals (49 ± 2 mg/ml). Blood samples were not collected during local perfusion with PGE1 but we assumed no systemic effects of the small amounts of PGE1 administered through the probe during the experiment. As expected, injection of 20 ml RS caused a large dilution of plasma, resulting in a decrease in Cp of all the EPP. During these experiments, blood samples were frequently collected, and the reduction was taken into account as previously described (Eq. 6).

If the increase in EPP or 125I-HSA concentration in the dialysate following docetaxel, PGE1, and RS was either entirely or partly due to changes in interstitial transport rates, criteria 2 would not be fulfilled. The relative recovery of the microdialysis probes would be subjected to variation during the experiment, and this effect would lead to an erroneous estimation of concentration changes in the interstitium. To compensate for any possible contribution from this effect, future studies should be performed with simultaneously retrodialysis with radiolabeled plasma proteins of known size during the experiment (22). This method involves measuring the loss of 125I- or 131I-labeled plasma proteins added to the perfusate to quantify variation in recovery due to changes in the organization of the extracellular matrix induced by the experimental protocol.

The large difference in albumin concentration in the dialysate collected from different probes during the baseline period (0–40 min, Table 1) indicates a large variance in recovery between probes. It was not unusual that the baseline concentration of albumin in two probes placed on the same animal differed from each other more than 100% (data not shown). Therefore, each probe served as its own control. The lack of steady state in our experiments, combined with the lack of conclusive support for the achievement of filtration rate independence, and lack of evidence of a constant recovery during the experiment, excluded the calculation of a value for {sigma}.

In conclusion, the microdialysis technique in its present form was not suitable to evaluate acute changes in capillary permeability. Also, the determination of EPP in interstitial fluid should be interpreted with caution due to the low recovery. However, the different pattern of extravasation of EPP and 125I-HSA after docetaxel and PGE1 indicates an effect on either or both the capillary wall and/or the interstitium for docetaxel, whereas PGE1 seems to increase Jv. In future studies, retrodialysis or other calibrating techniques (8) must be utilized to measure in situ changes in probe recovery. In addition, increased measurement time must be used to establish a steady-state between plasma, interstitium, and the microdialysis probe.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The present study received financial support from the Norwegian Health Association, The Norwegian Research council and Locus on Circulatory Research.


   ACKNOWLEDGMENTS
 
The technical assistance by Gerd Signe Salvesen is greatly appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Å. Borge, Dept. of Biomedicine, Section of Physiology, Univ. of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway (email: bengt.borge{at}biomed.uib.no)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aukland K and Fadnes HO. Protein concentration of interstitial fluid collected from rat skin by a wick method. Acta Physiol Scand 88: 350–358, 1973.[Web of Science][Medline]
  2. Aukland K and Reed RK. Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol Rev 73: 1–78, 1993.[Abstract/Free Full Text]
  3. Berg A, Ekwall AK, Rubin K, Stjernschantz J, and Reed RK. Effect of PGE1, PGI2, and PGF2{alpha} analogs on collagen gel compaction in vitro and interstitial pressure in vivo. Am J Physiol Heart Circ Physiol 274: H663–H671, 1998.[Abstract/Free Full Text]
  4. Brace RA, Granger DN, and Taylor AE. Analysis of lymphatic protein flux data III. Use of the nonlinear flux equation to estimate sigma and PS. Microvasc Res 16: 297–303, 1978.[CrossRef][Web of Science][Medline]
  5. Bronstad A, Berg A, and Reed RK. Effects of the taxanes paclitaxel and docetaxel on edema formation and interstitial fluid pressure. Am J Physiol Heart Circ Physiol 287: H963–H968, 2004.[Abstract/Free Full Text]
  6. Carter RD, Joyner WL, and Renkin EM. Effects of histamine and some other substances on molecular selectivity of the capillary wall to plasma proteins and dextran. Microvasc Res 7: 31–48, 1974.[CrossRef][Web of Science][Medline]
  7. Clough GF, Boutsiouki P, Church MK, and Michel CC. Effects of blood flow on the in vivo recovery of a small diffusible molecule by microdialysis in human skin. J Pharmacol Exp Ther 302: 681–686, 2002.[Abstract/Free Full Text]
  8. Desvigne N, Barthelemy JC, Bertholon F, Gay-Montchamp JP, Freyssenet D, and Costes F. Validation of a new calibration method for human muscle microdialysis at rest and during exercise. Eur J Appl Physiol 92: 312–320, 2004.[Web of Science][Medline]
  9. Dykstra KH, Hsiao JK, Morrison PF, Bungay PM, Mefford IN, Scully MM, and Dedrick RL. Quantitative examination of tissue concentration profiles associated with microdialysis. J Neurochem 58: 931–940, 1992.[Web of Science][Medline]
  10. Fadnes HO and Aukland K. Protein concentration and colloid osmotic pressure of interstitial fluid collected by the wick technique: analysis and evaluation of the method. Microvasc Res 14: 11–25, 1977.[CrossRef][Web of Science][Medline]
  11. Garlick DG and Renkin EM. Transport of large molecules from plasma to interstitial fluid and lymph in dogs. Am J Physiol 219: 1595–1605, 1970.[Free Full Text]
  12. Granger DN and Taylor AE. Permeability of intestinal capillaries to endogenous macromolecules. Am J Physiol Heart Circ Physiol 238: H457–H464, 1980.[Abstract/Free Full Text]
  13. Granger H, Dahr J, and Chen HI. Structure and function of the interstitium. In: Proceedings of the Workshop on Albumin, edited by Sgouris JTaR, A. Washington DC: Printing Office, 1975, p. 114–125.
  14. Groth L, Jorgensen A, and Serup J. Cutaneous microdialysis in the rat: insertion trauma studied by ultrasound imaging. Acta Derm Venereol 78: 10–14, 1998.[CrossRef][Web of Science][Medline]
  15. Groth L and Serup J. Cutaneous microdialysis in man: effects of needle insertion trauma and anaesthesia on skin perfusion, erythema and skin thickness. Acta Derm Venereol 78: 5–9, 1998.[CrossRef][Web of Science][Medline]
  16. Gyenge CC, Bowen BD, Reed RK, and Bert JL. Transport of fluid and solutes in the body II. Model validation and implications. Am J Physiol Heart Circ Physiol 277: H1228–H1240, 1999.[Abstract/Free Full Text]
  17. Iversen VV, Bronstad A, Gjerde EA, and Reed RK. Continuous measurements of plasma protein extravasation with microdialysis after various inflammatory challenges in rat and mouse skin. Am J Physiol Heart Circ Physiol 286: H108–H112, 2004.[Abstract/Free Full Text]
  18. Iversen VV and Reed RK. PGE1 induced transcapillary transport of 51Cr-EDTA in rat skin measured by microdialysis. Acta Physiol Scand 176: 269–274, 2002.[CrossRef][Web of Science][Medline]
  19. Joyner WL. Effect of prostaglandins on macromolecular transport from blood to lymph in the dog. Am J Physiol Heart Circ Physiol 232: H690–H696, 1977.[Abstract/Free Full Text]
  20. Joyner WL, Carter RD, Raizes GS, and Renkin EM. Influence of histamine and some other substances on blood-lymph transport of plasma protein and dextran in the dog paw. Microvasc Res 7: 19–30, 1974.[CrossRef][Web of Science][Medline]
  21. Joyner WL, Carter RD, and Renkin EM. Influence of lymph flow rate on concentrations of proteins and dextran in dog leg lymph. Lymphology 6: 181–186, 1973.[Web of Science][Medline]
  22. Larsson CI. The use of an "internal standard" for control of the recovery in microdialysis. Life Sci 49: PL73–78, 1991.[CrossRef][Web of Science][Medline]
  23. Lumley P, Humphrey PP, Kennedy I, and Coleman RA. Comparison of the potencies of some prostaglandins as vasodilators in three vascular beds of the anaesthetised dog. Eur J Pharmacol 81: 421–430, 1982.[CrossRef][Web of Science][Medline]
  24. Manning RD Jr and Guyton AC. Dynamics of fluid distribution between the blood and interstitium during overhydration. Am J Physiol Heart Circ Physiol 238: H645–H651, 1980.[Abstract/Free Full Text]
  25. Mullins RJ and Bell DR. Changes in interstitial volume and masses of albumin and IgG in rabbit skin and skeletal muscle after saline volume loading. Circ Res 51: 305–313, 1982.[Free Full Text]
  26. Mullins RJ and Bell DR. Permeability of rabbit skin and muscle microvasculature after saline infusion. J Surg Res 32: 390–400, 1982.[CrossRef][Web of Science][Medline]
  27. Owen M and Triffitt JT. Extravascular albumin in bone tissue. J Physiol 257: 293–307, 1976.[Abstract/Free Full Text]
  28. Patlak CS, Goldstein DA, and Hoffman JF. The flow of solute and solvent across a two-membrane system. J Theor Biol 5: 426–442, 1963.[CrossRef][Web of Science][Medline]
  29. Reed RK. Transcapillary albumin extravasation in rat skin and skeletal muscle: effect of increased venous pressure. Acta Physiol Scand 134: 375–382, 1988.[Web of Science][Medline]
  30. Renkin EM, Gustafson-Sgro M, and Sibley L. Coupling of albumin flux to volume flow in skin and muscles of anesthetized rats. Am J Physiol Heart Circ Physiol 255: H458–H466, 1988.[Abstract/Free Full Text]
  31. Renkin EM, Joyner WL, Sloop CH, and Watson PD. Influence of venous pressure on plasma-lymph transport in the dog's paw: convective and dissipative mechanisms. Microvasc Res 14: 191–204, 1977.[CrossRef][Web of Science][Medline]
  32. Renkin EM, Rew K, Wong M, O'Loughlin D, and Sibley L. Influence of saline infusion on blood-tissue albumin transport. Am J Physiol Heart Circ Physiol 257: H525–H533, 1989.[Abstract/Free Full Text]
  33. Renkin EM, Tucker V, Rew K, O'Loughlin D, Wong M, and Sibley L. Plasma volume expansion with colloids increases blood-tissue albumin transport. Am J Physiol Heart Circ Physiol 262: H1054–H1067, 1992.[Abstract/Free Full Text]
  34. Rippe B, Haraldsson B, and Folkow B. Evaluation of the "stretched pore phenomenon" in isolated rat hindquarters. Acta Physiol Scand 125: 453–459, 1985.[Web of Science][Medline]
  35. Rutili G, Granger DN, Taylor AE, Parker JC, and Mortillaro NA. Analysis of lymphatic protein data. IV Comparison of the different methods used to estimate reflection coefficients and permeability-surface area products. Microvasc Res 23: 347–360, 1982.[CrossRef][Web of Science][Medline]
  36. Schmelz M, Luz O, Averbeck B, and Bickel A. Plasma extravasation and neuropeptide release in human skin as measured by intradermal microdialysis. Neurosci Lett 230: 117–120, 1997.[CrossRef][Web of Science][Medline]
  37. Semb KA, Aamdal S, and Oian P. Capillary protein leak syndrome appears to explain fluid retention in cancer patients who receive docetaxel treatment. J Clin Oncol 16: 3426–3432, 1998.[Abstract]
  38. Simchon S, Carlin RD, Jan KM, and Chien S. A double isotope technique to determine regional albumin permeability: effects of anesthesia. Proc Soc Exp Biol Med 195: 114–118, 1990.[Abstract]
  39. Taylor A and Granger G. Exchange of macromolecular substances across the capillary wal. In: Handbook of Physiology. The Cardiovascular system. Microcirculation. Bethesda, MD: Am. Physiol. Soc., 1984, sect. 2, vol. IV, pt. 1, chapt. 11, p. 467–520.
  40. Wallace JR and Bell DR. Comparison of protein lymph flux and extravascular uptake in skin during increased venous pressure. Am J Physiol Heart Circ Physiol 263: H895–H902, 1992.[Abstract/Free Full Text]
  41. Wiig H, Aukland K, and Tenstad O. Isolation of interstitial fluid from rat mammary tumors by a centrifugation method. Am J Physiol Heart Circ Physiol 284: H416–H424, 2003.[Abstract/Free Full Text]
  42. Wiig H, Heir S, and Aukland K. Colloid osmotic pressure of interstitial fluid in rat subcutis and skeletal muscle: comparison of various wick sampling techniques. Acta Physiol Scand 133: 167–175, 1988.[Web of Science][Medline]
  43. Wolf MB, Scott DR, II, and Watson PD. Microvascular permeability transients due to histamine in cat limb. Am J Physiol Heart Circ Physiol 261: H220–H228, 1991.[Abstract/Free Full Text]




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