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Department of Physiology, University of Bergen, N-5009 Bergen, Norway
Submitted 24 June 2003 ; accepted in final form 21 August 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inflammation; permeability; microcirculation
Microdialysis with large-pore-size membranes has been used successfully to investigate neuropeptide-induced inflammation and PPE in human skin in vivo (16). First, this technique has the advantage of being relatively atraumatic. Second, it is possible to deliver large-sized molecular substances locally to the tissue via the fiber. Finally, the method allows continuous measurement of the response with simultaneous administration of the treatment. Acute inflammation in skin is accompanied by increased negativity of Pif, which increases capillary fluid filtration and thereby potentiates edema formation (12). Our group has over the years studied the effect of several inflammatory mediators on Pif with the micropuncture technique. Microdialysis complements these studies by measuring PPE. We previously measured (8) capillary-to-interstitium water transport after PGE1 by using microdialysis and 51Cr-EDTA as a tracer.
Compared with lymphatic and interstitial protein concentration for determination of capillary permeability, the microdialysis method has the advantage over lymphatic sampling that the sampling site is much closer to the capillary wall. This markedly reduces the time required to obtain a steady state. Also, the method has the advantage over isotope studies 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 blood-to-tissue exchange with radioactive tracers is used.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals were obtained from M & B. They were fed a standard diet and tap water ad libitum and housed under conventional conditions. Female Wistar rats (200–250 g) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip, with supplemental doses administered when required). Female C57 black mice were anesthetized at a volume of 0.1 ml/10 g body wt subcutaneously, with supplemental doses administered when required, of a 1:1:2 mixture of Hypnorm (fentanyl-fluanison), midazolam (5 mg/l), and distilled water. Cannulation of the left femoral vein was performed for intravenous injections of 125I-labeled human serum albumin (HSA), dextran, and phalloidin. The experiments in this study were performed with the approval of, and in accordance with the regulations laid down by, the Norwegian State Commission for Laboratory Animals.
Microdialysis
Microdialysis was performed as described by Schmelz et al. (16). Hollow plasmapheresis fibers (0.4-mm diameter, cutoff 3,000 kDa; PF2000, Gambro) were glued (Scotch superglue) to plastic tubing (PE-50) with an active membrane of 3 cm. Two microdialysis fibers were placed subcutaneously: longitudinally on both sides of the spine of mice and transversally on the back of rats with a distance of 4 cm between the fibers. The fibers were perfused with 0.9% NaCl by a microdialysis pump (CMA/100) at a constant flow rate of 5 µl/min. The dialysate was collected after passage through the skin at 10-min intervals from 6-cm-long outlet tubing inserted in plastic vials (Eppendorf). After a stabilization period of 30 min, a baseline period of 30–40 min commenced. After the baseline period, the effects of the substances were recorded for 70 min: either dextran or phalloidin (iv; both as bolus injection) or PGE1, docetaxel, paclitaxel, Cremophor EL, or compound 48/80 was given continuously via the fiber. In the experiments in which the substance was given via the fiber, a control fiber was perfused with saline during the whole experiment. Capillary-to-interstitium transport of protein by microdialysis after intravenous injection of 125I-HSA (Institute For Energy Technique, Kjeller, Norway) was determined in a gamma counting system (LKB Wallac, Turku, Finland) with automatic background and spillover correction. The stock solution of 125I-HSA contained <3% free iodide according to the manufacturer.
Radioactivity in the baseline period (30–40 min) was set as the basal extravasation of protein. Experimental values after injection of the active substance were calculated as a percentage of baseline. In vitro recovery for 125I-HSA was measured by perfusing single hollow fibers at 5 µl/min placed in a saline containing 125I-HSA and with an active microdialysis membrane of 3 cm as in the in vivo studies. The in vitro recovery of the fibers was found to be 45 ± 10% when placed in aqueous solution.
Experimental Protocol
After the surgical procedures and implantation of the microdialysis fibers were completed, the animal was left to stabilize for 30 min with the fibers being perfused with saline at a rate of 5 µl/min. The animals then received 0.2 ml of 125I-HSA (10 MBq) intravenously circulated for another 10 min. Sampling for the subsequent 30–40 min represented a baseline with collection of the dialysate in sampling vials every 10 min. After the baseline period, tubing was swapped from syringes containing saline to syringes containing the substance under study by a liquid switch (CMA/110) in the experiments in which the substance was delivered via the fiber. In the experiments in which the substance was given intravenously there was no swapping of syringes. Sampling of dialysate progressed for 70 min with collection of the dialysate every 10 min.
Experimental Groups
Dextran. Dextran (0.2 ml) was injected intravenously (Macrodex; 60 mg/ml) in six rats with ligated kidneys and four rats with nonligated kidneys.
Compound 48/80. Compound 48/80 (Sigma, St. Louis, MO) was used at 4 mg/ml and was given via the microdialysis fiber (n = 6).
Prostaglandin E1. PGE1 (Sigma) was used at 20 µg/ml and given via the microdialysis fiber (n = 6).
Paclitaxel. Paclitaxel (Taxol, paclitaxel in ethanol and its solvent Cremophor EL; Bristol-Myers Squibb) was supplied at 6 mg/ml and used at 0.6 mg/ml after dilution in sterile saline (0.9% NaCl) and was given via the microdialysis fiber (n = 5).
Docetaxel. Docetaxel (Taxotere, docetaxel 40 mg/ml, Aventis Pharma) was diluted to concentration of 0.5 mg/ml. The solution was used within 8 h after dilution and was given via the microdialysis fiber (n = 5).
Phalloidin. Phalloidin (Sigma) was dissolved in sterile saline (0.9% NaCl) to a final concentration of 1 mg/ml and was given at a dose of 500 µg/kg and administered 40 min before the second agent studied. One group of mice also received pretreatment with 50 µg/kg phalloidin before compound 48/80.
Statistical Methods
One-way ANOVA and subsequent Bonferroni post hoc tests were used. P < 0.05 was considered statistically significant. Values are means ± SE of percent increase compared with baseline.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fibers perfused with saline over a period of 30–40 min (baseline) plus 70 min (experimental) showed no change in PPE over the entire period compared with baseline (set as 100%). Pretreatment with phalloidin (n = 5) or Cremophor EL (n = 5) did not increase PPE during the experiment (Figs. 1 and 2).
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Experimental Groups
Experiments in rats. Intravenous injection of 0.2 ml dextran (60 mg/ml) increased PPE significantly by 355% (±59.7% SE) in rats with ligated kidneys compared with baseline after 30 min (the period of maximal radioactivity in the sample) (n = 6; P < 0.05), whereas it did not alter PPE from baseline in rats with nonligated kidneys (n = 4) (Fig. 3). The increase observed after intravenous injection of dextran was not significantly reduced in animals pretreated with phalloidin (500 µg/kg iv) 40 min before dextran when the kidneys were ligated. (n = 6; P < 0.05). However, when the kidneys were intact the increase in PPE after dextran was significantly reduced (P < 0.05; Fig. 3).
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PGE1 (15 µg/ml) given via the fiber increased PPE significantly by 139% (±73% SE), and maximal radioactivity in the sample was seen after 40 min (n = 6; P < 0.05). Pretreatment with phalloidin before PGE1 did not significantly inhibit the increase in PPE by PGE1 (n = 6; P < 0.05; Fig. 4).
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Docetaxel (0.5 mg/ml) increased PPE significantly compared with baseline within 60 min by 325%(±75.4% SE; n = 5; P < 0.05). Pretreatment with phalloidin before docetaxel significantly inhibited the increase in PPE (n = 6; P < 0.05; Fig. 5). Paclitaxel did not change PPE significantly compared with control (n = 5).
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Experiments in mice. Compound 48/80 significantly increased PPE by 273% (±55.6% SE), and maximum radioactivity in the sample was seen after 60 min (n = 6; P < 0.05; Fig. 6). Pretreatment with 500 µg/kg and 50 µg/kg phalloidin did not significantly inhibit the increase in PPE after compound 48/80.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study demonstrated that the microdialysis technique is suitable for measurement of PPE relatively atraumatically and continuously over a long period of time and with the immediate advantage of dramatically reducing the number of animals when measuring PPE at different time intervals, compared with timed sampling. The control groups (NaCl, phalloidin and Cremophor EL) had stable PPE at baseline level over the entire length of the experiments.
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. Direct comparison of the control and experimental fibers will yield the effect, which can be directly ascribed to the experimental maneuver.
Two different routes of administration were used. Dextran and phalloidin were given intravenously, whereas the other substances were given via the fiber. Local administration via the fiber was shown previously to be the preferred route of administration rather than local administration in bolus injections around the fiber (8), which leads to several unfavorable effects. Administration by local injection increases Pif and lower colloid osmotic pressure, which both lower capillary filtration. The increased fluid volume will also increase the diffusion length between the capillaries and the microdialysis fiber. The use of the fiber to administer the substance in question prevents this.
Trauma from the inflammation and possibly a low-grade inflammatory response is likely to result from the implantation of the microdialysis fiber. However, this will be the same for the control fiber and for the experimental fiber. Thus subtraction of the response in the control fiber from that in the experimental fiber will yield the biological response elicited by the agent studied. However, because of a potential presence and effect of a low-grade inflammation in the control fiber, care should be taken at present to use the microdialysis method to determine the "normal" extravasation of plasma proteins in an undisturbed and unperturbed state.
The substances investigated here have an effect at both the capillary level and the interstitial level. In the present experimental situation, it is the combined effect of these two levels that was recorded and measured in the microdialysate. Thus the deduction of what occurs at the capillary level also requires the knowledge of their combined effect and separate data for either the capillary or the interstitial effects of the agent. The interstitial effect has been studied fairly extensively with a protocol implying circulatory arrest followed by immediate interstitial administration of the agent under study. Alternatively, the agent has been administered by the intravenous route and shortly thereafter (minutes) circulatory arrest has been induced to avoid the inflammatory effects in the microcirculation, i.e., vasodilation, increased capillary pressure, and increased permeability for water and protein. In our previous studies (4, 5), we were only able to obtain a single point in the time course of capillary extravasation of albumin, whereas the present method permits determination of the whole time course of the transcapillary albumin flux. In this measurement, the current method has the advantage over the traditional lymphatic sampling and determination of protein concentration that the interstitial steady state required for the lymphatic approach is much less of a problem, if any at all. Compared with the isotope method using plasma-to-tissue clearance and subsequent tissue sampling, the current method has the advantage of allowing determination of the time course of the flux rather than a single point. On the other hand, the use of the microdialysis method with 125I-HSA does not allow determination of the increase in the transcapillary fluid flux, which, through the use of biopsies, is approximated by the increase in total tissue water.
In previous studies on rats, phalloidin attenuated the response on PPE induced by dextran but not PGE1 (Ref. 5; A. Brønstad, unpublished observations). This is in agreement with the trend in the present data, although a significant difference was not obtained. Also, in the present experiments there is some (statistically insignificant) lowering of PPE induced by phalloidin before PGE1, in agreement with the observation that, although phalloidin abolished lowering of Pif induced by PGE1, there was little effect on transcapillary albumin flux (A. Brønstad, unpublished observations). Similarly, the effects of dextran and docetaxel were more distinct also on the PPE, attesting to an effect both on the capillary and interstitial level. Furthermore, the time course of PPE is different with the three substances and in rats, with a marked peak after dextran at four times baseline, a doubling with a plateau after PGE1, and a constant increase during the period of observation after docetaxel. Finally, it should be noted that the increase in PPE is completely abolished after pretreatment with phalloidin. Together, the diverse effects of phalloidin on the increase in PPE induced by dextran, docetaxel, and PGE1 strongly suggest that these three agents have different sites of actions with regard to their effect on connective tissue cells and capillary endothelial cells. In mice, after compound 48/80 there is seemingly the opposite effect in that phalloidin in a seemingly dose-independent manner raises PPE above that seen with compound 48/80 alone but does not reach statistical significance.
Acute inflammation is associated with increased transcapillary flux and formation of edema (2, 14). We previously demonstrated (13) that the lowering of Pif seen in acute inflammation contributes to edema formation. It is believed that this is regulated by 
-integrin-mediated interactions between the connective tissue cells and the extracellular matrix. Blockade of -integrin function causes lowering of Pif and edema (13), and it is believed that the connective tissue cells actively contribute to the regulation of Pif. -Integrins are heterodimeric transmembrane receptors, which attach the cells to the extracellular matrix. The cell cytoskeleton is important for integrin function. Modulation of cell cytoskeleton function can modulate Pif and therefore the formation of edema. (5). Actin filaments are a group of cytoskeletal proteins that are important for locomotor functions of the cell and for the function of the -integrins. Actin filaments (F-actin) are built up by polymerization of globular actin (G-actin) units. Depolymerization of actin filaments with cytochalasin D lowers Pif and causes edema (4). Phalloidin from the mushroom Amanita phalloides stabilizes the actin filament system by inducing the assembly of F-actin (6, 17). It was shown previously that phalloidin attenuates inflammation induced by several inflammatory mediators as well as in ischemia-reperfusion injury (1, 11). Brønstad et al. (5) demonstrated that phalloidin pretreatment attenuated lowering of Pif, albumin extravasation and edema formation induced by dextran anaphylaxis, which is mediated by mast cell degranulation. The present data are in agreement with these studies.
We found an increase in PPE of 355% of baseline within 30 min after dextran in rats. For unexplained reasons pretreatment with phalloidin was only effective in rats when the kidneys were not ligated. When the kidneys were ligated, we observed the same PPE as in those treated only with dextran. Compound 48/80 administered to mice gave an increase of 273% of baseline. Pretreatment with phalloidin did not abolish this increase.
Microtubules, another group of cytoskeletal proteins, are built up by polymerization of tubulin monomers. Both actin filaments and microtubules are dynamic structures and are continuously reconstructed. The half-life of a microtubule is 
10 min. Paclitaxel and docetaxel belong to the taxanes and are derived from different yew trees (Taxus spp.). These substances are known to bind to the microtubules and thereby prevent depolymerization. Subdermal injection of paclitaxel and docetaxel caused lowering of Pif and edema (A. Brønstad, unpublished observations). Danowski (7) proposed a "push and pull theory" in which microtubulifixation causes reduced contractility of fibroblasts by opposing the contractile force induced by the actin filaments. This model would seem to explain the seeming contradiction that fixation of the three cytoskeletal structures caused opposite effects. Paclitaxel did not increase PPE. Docetaxel, however, increased PPE by 325% within 60 min. The lowering of Pif after docetaxel has been prevented by pretreatment with phalloidin (A. Brønstad, unpublished observations), which is in good agreement with the present study, because pretreatment with phalloidin completely abolished the increase in PPE observed after docetaxel alone. As discussed above, the effects of all substances tested here act at both the capillary and interstitial levels and it is their combined response that arises as PPE.
Cremophor EL is the solvent for paclitaxel. It has been suggested to have proinflammatory properties and cause adverse affects observed when paclitaxel (Taxol; 6 mg/ml) is used in cancer patients. Cremophor EL was tested on the effect on Pif and albumin extravasation in a previous study (A. Brønstad, unpublished observations). A similar conclusion was reached in this study, i.e., there was no effect above that of physiological saline.
PGE1 resulted in doubling of PPE within 60 min, and this increase was not attenuated by pretreatment with phalloidin, demonstrating that phalloidin does not prevent PPE induced by PGE1. This is in good agreement with a similar study (5) using the plasma clearance method with 25-min transcapillary extravasation of 125I-HSA and where phalloidin also failed to reduce edema formation. The results from the present study demonstrate that measurement of PPE with microdialysis is in good agreement with what has been shown in previous studies measuring either Pif, with the micropuncture method, or PPE, with the isotope-marked albumin clearance method.
In conclusion, the present study has demonstrated that the microdialysis technique can be used to measure increases in transcapillary transport of 125I-HSA in rat and mouse skin using this approach. The method gives an impression of the extravasation of plasma proteins over time and uses a small number of animals.
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ACKNOWLEDGMENTS |
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GRANTS
The present study received financial support from The Norwegian Heart Association and The Norwegian Research Council.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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analogs on collagen gel compaction in vitro and interstitial pressure in vivo. Am J Physiol Heart Circ Physiol 274: H663–H671, 1998.1-integrins. Semin Nephrol 21: 222–230, 2001.[CrossRef][ISI][Medline]
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