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THE EFFECT OF HYPOXIA, REOXYGENATION, ISCHEMIA, AND REPERFUSION ON HYDRAULIC PERMEABILITY IN RAT MESENTERIC VENULES

Victorino, Gregory P.; Chong, Terry J.; Cripps, Michael W.; Ereso, Alexander Q.; Cureton, Elizabeth; Curran, Brian; Sadjadi, Javid

doi: 10.1097/SHK.0b013e318183376c
Basic Science Aspects
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Little is known regarding the effects of I/R on hydraulic permeability (Lp). We sought to compare the individual influences of hypoxia, ischemia, reoxygenation, and reperfusion on Lp. We hypothesized that (1) hypoxia increases Lp; (2) reoxygenation further increases Lp; (3) ischemia results in greater increases in Lp compared with hypoxia; (4) reperfusion causes additional increases in Lp compared with hypoxia, ischemia, and reoxygenation; and (5) xanthine oxidase (XO) and white blood cell adherence play important roles in hypoxia, ischemia, and reperfusion. Hydraulic permeability was measured by an in vivo microcannulation technique during hypoxia, reoxygenation, ischemia, and reperfusion in rat mesenteric postcapillary venules. Additional rats were fed a Tungsten-enriched diet to inhibit XO activity, and the studies were repeated. White blood cell adherence was also documented. Hypoxia and ischemia both increased Lp 2-fold from baseline levels (P < 0.001). Reoxygenation did not alter Lp compared with 15 min of hypoxia alone (P > 0.07). Reperfusion after hypoxia increased Lp 6-fold (P < 0.001). Reperfusion after ischemia also increased Lp 6-fold (P < 0.001). Inhibition of XO had no effect on the increase in Lp after both hypoxia and ischemia. However, inhibition of XO attenuated the 6-fold increase in Lp observed during reperfusion after both hypoxia and ischemia by approximately 50% (P < 0.001). White blood cell adherence increased during reperfusion but not hypoxia or ischemia. The complexity of I/R injury makes it a difficult clinical scenario to model for research. We have demonstrated in an in vivo model that hypoxia and ischemia increase Lp similarly, and that reperfusion has a profound deleterious effect on Lp. These changes in Lp seem to be XO and white blood cell dependent.

University of California San Francisco East Bay Department of Surgery, Alameda County Medical Center, Oakland, California

Received 28 Apr 2008; first review completed 13 May 2008; accepted in final form 27 May 2008

Address reprint requests to Gregory P. Victorino, MD, Department of Surgery, UCSF-East Bay, 1411 East 31ST Street, Oakland, CA 94602. E-mail: victorinog@surgery.ucsf.edu.

This study was supported by the American Association for the Surgery of Trauma Research Scholarship Award.

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INTRODUCTION

Although the effects of I/R on cellular damage and macromolecular permeability have been well described, little is known regarding the effects on hydraulic conductivity (1-3). In I/R, most endothelial cell dysfunction occurs at the postcapillary venule. This dysfunction includes endothelial-leukocyte adhesion, increased oxidant production, platelet aggregation, paracellular leukocyte migration, and increased microvascular permeability (4). However, most of the data on microvascular permeability during oxidant stress come from cultured endothelial cell and monolayer studies, which, although valuable, do not account for the impact of in vivo physical qualities, including basement membrane-endothelial cell interaction, interstitial tissue effects, shear stress, and the influence of auxiliary cells and blood components (5). It is also difficult to model I/R injury using in vitro systems. There are in vivo evaluations of endothelial cell barrier dysfunction using intravital microscopic measurements of fluorescein isothiocyanate-albumin (6) or models that measure osmotic reflection coefficient to plasma protein (7), but to our knowledge, no one has measured hydraulic permeability (Lp) during I/R.

By measuring transendothelial electrical resistance or passage of radiolabeled macromolecules through cell monolayers, in vitro studies in cultured endothelial cells have shown that hypoxia alone induces barrier dysfunction (8, 9). In contrast, in vivo models have demonstrated that most endothelial barrier dysfunctions are attributed to the reperfusion of the tissue involved in I/R injury (10, 11). A previous investigation reported that trafficking of leukocytes was the rate-limiting step in I/R-induced barrier dysfunction, and that the amount of albumin leakage correlated with the number of adherent leukocytes during reperfusion (6). These disparities between the in vitro and in vivo studies prompted us to evaluate the effects of oxidant stress on Lp.

We sought to compare the differential influences of hypoxia, ischemia, reoxygenation, and reperfusion on hydraulic conductivity. The modified Landis micro-occlusion technique was used to determine Lp in rat mesenteric postcapillary venules. This is an ideal technique to measure Lp because it allows endothelial structural integrity to be maintained and auxiliary cells and other blood components to be preserved during reperfusion while controlling for surface area, hydrostatic pressure, and osmotic pressure. In this study, we investigated five hypotheses: (1) hypoxia alone increases Lp; (2) reoxygenation after hypoxia further increases Lp; (3) ischemia results in greater increases in Lp than hypoxia alone; (4) reperfusion after ischemia causes additional increases in Lp compared with hypoxia, ischemia, and reoxygenation; and (5) xanthine oxidase (XO) and white blood cell (WBC) adherence play important roles in hypoxia, ischemia, and reperfusion.

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MATERIALS AND METHODS

Animal and solution preparations

All studies were approved and complied with institutional animal research protocols. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. Preparation of the animals and preparation of the mammalian Ringer solution have been described previously (12). They are briefly described in the succeeding sentences.

Red blood cells that are used as flow markers were harvested from female Golden Syrian hamsters (140-180 g; Harlan, Indianapolis, Ind). The blood was centrifuged to remove the plasma and buffy coat and then washed three times in 15 mL of mammalian Ringer solution.

The Ringer solution was prepared daily in distilled deionized water and contained 135 mM NaCl, 4.6 mM KCl, 2.0 mM CaCl, 2.46 mM MgS04, 5.0 mM NaHCO3, 5.5 mM dextrose, 9.03 mM HEPES salt (Research Organics; Cleveland, OH), and 11.04 mM HEPES acid (Research Organics). Bovine serum albumin (BSA) solution was added to Ringer solution to prepare a 1% BSA Ringer solution for perfusion (BSA crystallized; Sigma Chemical, St. Louis, Mo).

Adult female Sprague-Dawley rats (250-310 g; Hilltop Lab Animals, Inc., Scottsdale, Pa) were anesthetized with subcutaneous sodium pentobarbital (60 mg/kg body weight). The bowel mesentery was gently exposed and positioned on an inverted microscope stage (Diaphot, Nikon; Melville, NY). The animal's body temperature was maintained at 37°C throughout the study. The mesentery was continuously bathed in Ringer solution.

Rat mesenteric postcapillary venules, 20 to 30 μm in diameter and at least 400 μm in length, were identified based on flow patterns. Vessels with no evidence of leukocyte adherence or side branches were chosen. The vessels were cannulated with micropipettes attached to a water manometer for control of hydrostatic perfusion pressure.

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Measurement of Lp

Single-vessel Lp was determined using the modified Landis micro-occlusion technique. The assumptions and limitations of this model have been previously described (13). Initial cell velocity (dl/dt) was obtained by recording marker cell position as a function of time. Transmural water flux per unit area (Jv/S) was calculated by the equation Jv/S = (dl/dt)(r/21), where r is the capillary radius and 1 is the initial distance between the marker cell and the occluded site. Determination of Lp was based on a modified version of the Starling equation of fluid filtration: Lp = (Jv/S) (1/Pc), where Pc is the capillary hydrostatic pressure. Hydraulic permeability was calculated from the slope of the regression of Jv/S on Pc derived from several occlusions at three different perfusion pressures. The units for Lp are cm·s−1·cmH2O−1 x 10−7. Control studies that document the stability of this model over time and after multiple recannulations of the vessels have been previously reported (12).

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Inhibition of XO

To assess the effect of XO inhibition on I/R and microvascular fluid leak, six rats were fed a Tungsten-enriched diet (modified AIN-93M purified rodent diet without molybdenum with 0.7 g/kg sodium tungstate dihydrate; Dyets, Inc., Bethlehem, Pa) ad libitum for 14 days before the I/R study. The addition of tungsten to a molybdenum-depleted diet causes the replacement of molybdenum as a coenzyme to XO. The replacement of molybdenum with tungsten leads to the inhibition of XO activity, which approaches zero after 14 days on this diet (14). After being on the tungsten-enriched diet for 2 weeks, rats then underwent the I/R protocol with serial measures of Lp as previously discussed.

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WBC adhesion

White cell adhesion was documented by intravital microscopy. White cells that were firmly adherent to the wall of the study vessel were assessed during videotape reply. White cell adherence was documented as the number of adherent white cells per 150 μM of study vessel.

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Hypoxia protocol

Hypoxia was induced by lowering the microvascular environment's atmospheric oxygen content from 21% to 10%. This corresponded to a drop in arterial PaO2 from 99 to 47 mmHg and a drop in arterial Sao2 from 97% to 85% (arterial blood gas analyzed by iStat Analyzer; iStat Corporation, East Windsor, NJ). First, the rat was placed in a sealed container infused with nitrogen gas to lower fractional inspired oxygen to 10%. Second, throughout the experiments, the superfusate that was used to bathe the rat mesentery was bubbled with nitrogen gas to lower oxygen content to 10%. Third, the perfusates in the pipettes that were used to cannulate the study venules were also made hypoxic by bubbling nitrogen gas into the solution before microvessel cannulation. Study venules were cannulated and perfused with the hypoxic Ringer solution. Oxygen probes (MI-730 micro-oxygen electrode; Microelectrodes, Inc., Bedford, NH) were used to ensure 10% atmospheric oxygen environment throughout the experiments. The oxygen probes were placed inside the sealed container, directly on the mesentery, and close to the study venule. This protocol thus allows for the measurements of the effects of hypoxia while still allowing perfusion of the vessels.

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Ischemia protocol

To make the study venules ischemic, the venules were made hypoxic as previously discussed. Then, the microvascular flow through the venule was stopped by obstructing inflow proximally via the cannulation micropipette and distally via a glass micro-occluder. This prevented any flow through the study vessel, which was confirmed by intravital microscopy.

The key difference between the hypoxia protocol and the ischemia protocol is that flow is present during the hypoxia protocol. The ischemia protocol provides a hypoxic environment while preventing flow through the vessels. This allows for the comparison of hypoxia versus ischemia.

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Reoxygenation protocol

Reoxygenation was accomplished by returning the hypoxic environment to normoxic levels (fractional oxygen content of 0.21). This included opening the sealed container to atmospheric oxygen, changing the hypoxic superfusate to a solution made under atmospheric conditions, and recannulating the venule and perfusing with a Ringer solution made under atmospheric conditions. Oxygen probes were used again to document that the oxygen content was 21%. This corresponded to an increase in arterial PaO2 from 47 to 99 mmHg and an improvement in arterial Sao2 from 85% to 96%.

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Reperfusion protocol

Reperfusion was achieved by allowing each rat's autologous blood to freely flow through the microvessel while simultaneously achieving reoxygenation as previously described. Autologous blood flow was reestablished by removing the cannulation micropipette and the glass micro-occluder from obstructing flow through the study venule. The reperfusion portion of the study allows exposure of the study venule to all the activated cellular elements and vasoactive mediators that are up-regulated during I/R.

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Experimental design

Effect of hypoxia on Lp

After initial measurements of Lp were made during baseline normoxic states, additional Lp measurements were obtained at 5-min intervals during a 40-min period of continuous hypoxia (n = 6).

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Effect of hypoxia and reoxygenation on Lp

After baseline measurements were made, Lp was determined during hypoxia for either 10 (n = 6) or 15 min (n = 6). Hydraulic permeability was measured again after reoxygenation at 5-min intervals for 20 min.

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Effect of hypoxia and reperfusion on Lp

After baseline Lp measurements were obtained, rats underwent hypoxia for 15 min, after which Lp was measured. Then, the study venules underwent reperfusion for 15 min, and Lp was measured again. Study vessels were than recannulated with normoxic Ringer, and Lp was measured at 5-min intervals during the final 20 min of the experiment (n = 6). White blood cell adherence was documented at baseline, after hypoxia, and after reperfusion.

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Effect of I/R on Lp

After baseline Lp was obtained, rats underwent ischemia for 15 min, after which Lp was measured again. The study venules then underwent reperfusion for 15 min, after which Lp was measured. Study vessels were then recannulated with normoxic mammalian Ringer, and Lp was measured at 5-min intervals during the final 20 min of the experiment (n = 6). White blood cell adherence was documented at baseline, after ischemia, and after reperfusion.

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Effect of XO inhibition on hypoxia, ischemia, and reperfusion on Lp

Rats were place on a tungsten-enriched diet to inhibit XO production. These animals then underwent the hypoxia reperfusion (n = 4) and the I/R (n = 4) protocols as previously discussed. White blood cell adherence was again documented at baseline, after hypoxia or ischemia, and after reperfusion.

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Statistics

Group means of sequential measurements were analyzed by repeated-measures ANOVA with post hoc analysis. Measurements between different study groups (hypoxia, hypoxia-reoxygenation, hypoxia-reperfusion, and I/R) were analyzed using unpaired Student t tests and ANOVA with post hoc analysis. Statistical significance was set at an α error of 5%. All values for Lp are represented as mean ± SEM × 10−7 cm·s−1·cm H2O−1.

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RESULTS

Effect of hypoxia on Lp

Hypoxia increased Lp steadily from a baseline of 1.16 ± 0.10 to a peak of 2.22 ± 0.23 at 15 min (P < 0.001; Fig. 1). Despite continuous hypoxia, Lp then decreased back to baseline levels by 30 min and remained at baseline levels for the remainder of the 40-min study period.

Fig. 1

Fig. 1

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Effect of hypoxia and reoxygenation on Lp

After an identical 2-fold increase in Lp with 15 min of hypoxia from a baseline of 0.92 ± 0.07 to a peak of 1.98 ± 0.04 (P < 0.001), reoxygenation did not alter Lp compared with continuous hypoxia alone at all time points (P > 0.07; Fig. 2). With hypoxia-reoxygenation, the Lp peaked at 15 min and returned to baseline levels by 25 min, which was similar to continuous hypoxia alone.

Fig. 2

Fig. 2

In contrast, reoxygenating after only 10 min of hypoxia did not reach the same peak Lp as compared with continuous hypoxia alone (Fig. 2). Compared with a peak Lp of 2.22 ± 0.23 at 15 min of continuous hypoxia, the Lp during reoxygenation after 10 min of hypoxia reached a peak of only 1.80 ± 0.12, and the Lp decreased to 1.54 ± 0.10 by the 15-min time point (P < 0.05). The Lp peaked at 10 min, which was less than a 2-fold increase from baseline (baseline Lp, 1.21 ± 0.03; peak Lp, 1.80 ± 0.12) and then declined back toward baseline levels.

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Effect of hypoxia and reperfusion on Lp

Once again, 15 min of hypoxia increased Lp by 2-fold from a baseline of 1.07 ± 0.13 to 2.18 ± 0.23 (P < 0.001; Fig. 3). However, unlike continuous hypoxia and hypoxia-reoxygenation, both of which subsequently decreased Lp to baseline levels, reperfusion increased Lp further to 6.21 ± 0.20 at the end of 15 min of reperfusion.

Fig. 3

Fig. 3

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Effect of I/R on Lp

Ischemia for 15 min increased Lp by approximately 2-fold from a baseline of 1.05 ± 0.05 to 2.53 ± 0.11 (P < 0.001), whereas reperfusion further increased Lp by more than 3-fold to 7.07 ± 0.30 (P < 0.001). These results are not significantly different from hypoxia-reperfusion; the line graphs for I/R and hypoxia reperfusion almost overlap in Figure 3 (P > 0.06).

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Effect of XO inhibition on hypoxia, ischemia, and reperfusion on Lp

Inhibition of XO by placing rats on a tungsten-enriched diet had no effect on the 2-fold increase in Lp that was observed after both hypoxia and ischemia (P = 0.2; Fig. 4). However, compared with hypoxia-reperfusion alone, XO inhibition attenuated the 6-fold increase in Lp observed during reperfusion after hypoxia by 48%, from an Lp of 6.21 ± 0.20 down to 3.26 ± 0.16 (P < 0.001). Compared with I/R alone, XO inhibition also attenuated the 6-fold increase in Lp observed during reperfusion after ischemia by 47%, from an Lp of 7.07 ± 0.30 down to 3.78 ± 0.24 (P < 0.001).

Fig. 4

Fig. 4

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WBC adherence during hypoxia, ischemia, and reperfusion

White blood cell adherence was similar in all groups at baseline (mean, 5.05 ± 1.9 adherent WBC per 150 μM of vessel; Fig. 5). This was unchanged after 15 min of hypoxia or ischemia in rats on a regular diet and also rats on the tungsten-enriched diet (mean, 7.5 ± 2.1 adherent WBC per 150 μM of vessel; P = 0.08). However, WBC adherence increased after 15 min of reperfusion in all four groups. After hypoxia-reperfusion, WBC adherence increased 5-fold (to 31.3 ± 1.8; P < 0.001), and this was attenuated 31% due to XO inhibition (P < 0.01). After I/R, WBC adherence increased 3.7-fold (to 34.3 ± 2.1; P < 0.001), and this was attenuated 18% due to XO inhibition (P < 0.03).

Fig. 5

Fig. 5

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DISCUSSION

Several in vitro studies demonstrate that hypoxia increases endothelial cell damage and monolayer macromolecule permeability in bovine, murine, and human endothelial cell lines (1, 2, 8, 9). In vivo models have confirmed the cell culture studies and have demonstrated that there is increased leukocyte rolling/adherence and microvascular permeability after hypoxia (15, 16). I/R models have also shown increases in leukocyte adhesion and protein leakage from the microvasculature (6). However, changes in Lp have not been evaluated in an in vivo hypoxia-reoxygenation, hypoxia-reperfusion, and I/R model. Our in vivo studies demonstrated that (1) hypoxia transiently increased Lp, (2) reoxygenation did not exacerbate Lp after hypoxia, (3) ischemia and hypoxia elevated Lp in similar magnitudes, (4) reperfusion after either hypoxia or ischemia caused additional increases in Lp, and (5) XO and WBC adherence play important roles in hypoxia, ischemia, and reperfusion.

During continuous hypoxia, we found that Lp transiently increased by 2-fold at the 15-min time point. Despite continued hypoxia for an additional 25 min, the Lp returned to and remained at baseline levels. Others have shown that hypoxia increases the permeability to macromolecules in bovine pulmonary and aortic endothelial cell monolayers (9). In addition, consistent with our results is an in vivo study that demonstrated similar transient increases in hydraulic conductivity in amphibian mesenteric venules exposed to hypoxia (15).

Interestingly, and counter to our hypothesis, after 15 min of hypoxia, reoxygenation did not further increase Lp. However, when the postcapillary venule was exposed to the hypoxic environment for a shorter time period (10 min), there was less of a response because the peak Lp did not reach the same levels as it did after hypoxia for 15 min. Reoxygenation does not seem to have a detrimental effect on Lp, and reoxygenating sooner may prevent the deleterious effects of hypoxia. This lack of detrimental effect disproves our hypothesis that reoxygenation would cause additional increases in Lp due to augmented oxygen free-radical production by providing a greater supply of oxygen. Earlier studies on hypoxia-reoxygenation have demonstrated injury or permeability changes on endothelial cells. Although these studies showed an in increase in endothelial cell permeability caused by increased generation of oxygen radicals, they demonstrated the effect after hypoxia followed by reoxygenation, rather than measuring the effect first after hypoxia and again after reoxygenation (1, 2, 17). Therefore, it is difficult to differentiate the individual effects of hypoxia and reoxygenation in these studies. In our model, we were able to separate the individual effect of each insult and were able to demonstrate the lack of an expected increase in Lp due to reoxygenation.

In our study, reperfusion had a strikingly different effect than reoxygenation. This finding emphasizes the importance of humoral and cellular influences on endothelial barrier function. Unlike reoxygenation, reperfusion increased Lp an additional 3-fold over the 2-fold increase from hypoxia or ischemia alone. Our results are supported by those from a study of intestinal microvascular macropermeability represented by the osmotic reflection coefficient. In this study, microvascular macromolecule permeability increased during ischemia and increased further when ischemia was combined with reperfusion (18). The reexposure to auxiliary cells and other blood components demonstrated a dramatic elevation of Lp in our model, which shows their significant role in increasing fluid leak from postcapillary venules. This finding is in agreement with previous studies that have shown increased endothelial cell injury from extracellular production of superoxide radicals, whereas antioxidants such as superoxide dismutase, catalase, and allopurinol and mast cell stabilizers such as ketotifen prevented leukocyte adherence and endothelial cell injury (1, 2, 19).

In addition, counter to our hypothesis, we found that the effects of hypoxia and ischemia on Lp were similar. Measurements of Lp after I/R also showed no difference when compared with hypoxia-reperfusion. In our model, increases in Lp seemed to be more affected by hypoxia than by cessation of microvascular flow. The technique we used washed out WBCs and other blood components during the hypoxia and ischemia phases, so that these confounding factors were removed from the system. This allowed us to study the effects of microvascular flow and shear stress on hydraulic permeability without the additional effects from blood components. This technique may account for the discrepancy between our study and others that have shown flow rate significantly affecting leukocyte adherence and hydraulic conductivity (19, 20). The agents responsible for early I/R injury such as formation of reactive oxygen species, activation of preformed mediators, and membrane/protein damage from reactive oxygen species, may be the dominant factors responsible for the increase in hydraulic permeability in postcapillary venules, rather than the effects of flow versus no flow.

In conclusion, the complexity of I/R injury makes it a difficult situation to model for research. Cell culture and monolayer studies leave much unexplained. Our in vivo study was designed to separate many of the confounding factors that can affect endothelial barrier function during oxidant stress. We found that hypoxia and ischemia increase Lp similarly. At this early stage of I/R injury, the lack of oxygen may be the more important factor, rather than the lack of microvascular flow. The deleterious effect of reperfusion was confirmed by a further increase in Lp that was 3-fold higher than the levels observed after hypoxia or ischemia. Although these experiments were performed in rat mesenteric venules and are therefore not representative of the behavior of endothelial cells in other vessels and tissues, the results raise some interesting points. The pathologic process of I/R poses a difficult dilemma to physicians and is not confined to a specific field but is widely applicable to various disease processes in transplantation, cardiovascular disease, and sepsis/shock. Thus, there is a significant need for in vivo models that more accurately reflect this complex phenomenon.

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Keywords:

Microvascular permeability; hydraulic conductivity

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