Bronchial Microdialysis of Cytokines in the Epithelial Lining Fluid in Experimental Intestinal Ischemia and Reperfusion Before Onset of Manifest Lung Injury : Shock

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Bronchial Microdialysis of Cytokines in the Epithelial Lining Fluid in Experimental Intestinal Ischemia and Reperfusion Before Onset of Manifest Lung Injury

Tyvold, Stig Sverre*†; Solligård, Erik*†; Gunnes, Sigurd†‡; Lyng, Oddveig§; Johannisson, Anders∥; Grønbech, Jon E.§¶; Aadahl, Petter†‡

Author Information

*Department of Anesthesia and Intensive Care, St Olavs Hospital; †Department of Circulation and Medical Imaging, Norwegian University of Science and Technology; ‡Department of Heart and Lung Surgery, St Olavs Hospital; and §Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway; ∥Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; and Department of Gastrointestinal Surgery, St Olavs Hospital, Trondheim, Norway

Received 26 Feb 2010; first review completed 4 Mar 2010; accepted in final form 9 Mar 2010

Address reprint requests to Stig Sverre Tyvold, Department of Circulation and Medical Imaging, The Faculty of Medicine, MTFS, Norwegian University of Science and Technology, 7489 Trondheim, Norway. E-mail: [email protected].

This study was supported by grants from St Olavs Hospital, Helse Midt-Norge, and the Norwegian University of Science and Technology, Trondheim, Norway.

Shock 34(5):p 517-524, November 2010. | DOI: 10.1097/SHK.0b013e3181dfc430
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Abstract

Today, there is no continuous monitoring of the bronchial epithelial lining fluid. This study used microdialysis as a method of continuous monitoring of early lung cytokine response secondary to intestinal ischemia-reperfusion in pigs. The authors aimed to examine bronchial microdialysis for continuous monitoring of IL-1β, TNF-α, IL-8, and fluorescein isothiocyanate Dextran 4,000 Da (FD-4). The superior mesenteric artery was cross-clamped for 120 min followed by 240 min of reperfusion (ischemia group, n = 8). Four sham-operated pigs served as controls. The pigs were anesthetized and normoventilated (peak inspiratory pressure, <20 cm H2O; positive end-expiratory pressure, 7 cm H2O). Samples from bronchial and luminal intestinal and arterial microdialysis catheters (flow-rate of 1 μL/min) were collected during reperfusion in 60-min fractions. Samples were analyzed for TNF-α, IL-1β, IL-8, and FD-4. Data are presented as median (interquartile range). A lung biopsy was collected at the end of the experiment. During reperfusion, there was an increase in bronchial concentrations of both IL-8 (3.70 [1.47-8.93] ng/mL per h vs. controls, 0.61 [0.47-0.91] ng/mL per h; P < 0.001) and IL-1β (0.32 [0.05-0.56] ng/mL per h vs. controls, 0.07 [0.04-0.10] ng/mL per h; P = 0.008). In the intestinal lumen, IL-8 was increased in the ischemia group (6.33 [3.13-9.23] ng/mL per h vs. controls, 0.89 [0.21-1.86] ng/mL per h; P < 0.001). The FD-4 did not differ between groups. Pulmonary vascular resistance and pulmonary shunt increased versus controls. During reperfusion, PaO2/FiO2 ratio decreased in the ischemia group. Histology was normal in both groups. Bronchial microdialysis detects altered levels of cytokines in the epithelial lining fluid and can be used for continuous monitoring of the immediate local lung cytokine response secondary to intestinal ischemia-reperfusion.

INTRODUCTION

There is an ongoing search for robust prognostic factors in patients at risk or in the early phase of acute lung injury (ALI) and adult respiratory distress syndrome (ARDS). Inflammatory markers, such as interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), and compositions of these predict a favorable or unfavorable outcome (1, 2). Choices of treatment strategy, especially protective ventilation strategies, affect composition of inflammatory marker molecules and patient outcome (3). Early detection of inflammatory markers can be of great value in guiding the treatment of patients at risk for developing ALI/ARDS.

Most clinical studies have used blood samples or bronchoalveolar fluid to measure inflammatory markers in ALI/ARDS and have concluded that the inflammatory process in ALI/ARDS is best monitored within the lung (1-4).

Bronchoalveolar fluid can be obtained by various techniques. Bronchoalveolar lavage is intermittent, invasive, and has a range of adverse effects, such as hypoxemia, changes in lung mechanics, transient pulmonary hypertension, and local and systemic inflammatory activation (5-7). Bronchial microsampling is a promising method but is discontinuous and demands repeated fiber-optic bronchoscopies. The probe diameter (1.1 mm or 1.9 mm) is more than twice the diameter of the microdialysis catheter membrane (0.5 mm) and confines bronchial microsampling to larger airways (8, 9). Exhaled breath condensates are diluted about 2,000- to 10,000-fold, and epithelial lining fluid is mixed with fluids from all parts of the airway (10). Because of these methodological problems, the dynamic alterations of cytokines immediately after a primary insult sufficient to induce secondary ALI/ARDS are not studied in detail.

Microdialysis is an established method for continuous measurement of cytokines (11, 12).

Bronchial microdialysis is potentially a direct continuous monitor of multiple cytokines and a minimally invasive technique. Bronchial microdialysis has previously been used to evaluate the pharmacokinetics of antibiotics in rat lungs (13). We have recently described bronchial microdialysis as a useful monitor of biomarkers in bronchial epithelial lining fluid and improved the method's accuracy with a correction factor for the relative area of semipermeable membrane in contact with the bronchial epithelial lining fluid caused by position and the respiratory cycle (9).

Intestinal ischemia-reperfusion injury induces mild controlled secondary lung injury (14, 15).

We hypothesize that bronchial microdialysis can identify the immediate inflammatory response in the bronchi as measured by IL-1β, IL-8, and TNF-α and monitor the time-to-time changes of these cytokines during the first 4 h of reperfusion after intestinal ischemia.

METHODS

Anesthesia and surgical preparation

After institutional approval, 12 pigs (weighing 22-30 kg; Norwegian Landrace 50%, Landrace Duroc 25%, Yorkshire 25%) were anesthetized (continuous infusion of fentanyl 20-30 μg/kg per h and ketamine HCl 8-12 mg/kg per h), tracheostomized, and ventilated (FiO2, 0.21). To avoid ventilation-induced lung injury, peak inspiratory pressures was less than 20 cm H2O, positive end-expiratory pressure was set at 7 cm H2O, and tidal volumes were 6 to 8 mL/kg. Alongside the 7.0 endotracheal tube for ventilation, two 4.0 endotracheal tubes were inserted into the trachea for later introduction of the microdialysis catheters. The 4.0 endotracheal tubes were sealed with caps to avoid air leakage. A pulmonary artery catheter (Swan-Ganz CCOmbo 7.5F; Edwards Lifesciences, Irvine, Calif) was introduced into the right jugular vein and a triple lumen catheter (Certofix-Trio S715; Braun, Melsungen, Germany) in the femoral artery for hemodynamic monitoring and blood samples (9). Fluid balance was obtained by an i.v. infusion of heated (37°C) Ringer acetate at 10 to 15 mL/kg per h throughout the experiment. Rectal temperature was maintained within the normal range (37°C-39.6°C) by a heating mattress and wrappings (16).

Experimental protocol

In all animals, a midline laparotomy was performed, an occluder was placed around the superior mesenteric artery, and an ultrasound transit time flow probe (6 mm) was placed distal to the occluder around the superior mesenteric artery to verify intestinal ischemia (17). In the ischemia group (n = 8), the superior mesenteric artery was cross-clamped for 120 min followed by 240 min of reperfusion. Sham-operated pigs (n = 4) served as controls. After surgical preparations, the animals were allowed to stabilize for 60 min before baseline (Fig. 1).

F1-13
Fig. 1:
Experimental protocol. The time line is separated into three periods: baseline (60 min), ischemia (120 min), and reperfusion (240 min). The fluorescein isothiocyanate Dextran infusion started 70 min before baseline with a bolus of 10 μg/kg for 10 min followed by a continuous i.v. infusion of 5 μg/kg per h (22). Microdialysis sampling periods are marked as boxes. Two fractions of plasma from the femoral artery and serum from the pulmonal artery were drawn and prepared every 60 min, marked by arrows. Blood gases from the femoral and the pulmonal arteries were collected every 30 min, marked by arrows.

All animals were killed with i.v. pentobarbital 100 mg/kg after the experiment.

Hemodynamic and respiratory measurements

The mean arterial pressure, mean pulmonary artery pressure, pulmonary capillary wedge pressure, heart rate, and cardiac output were measured at 30-min intervals. Pulmonary vascular resistance was calculated (18). Arterial and mixed venous blood gases were sampled every 30 min. The PaO2/FiO2 ratio and shunt were calculated (19).

Microdialysis

Two microdialysis catheters (custom-made, 10-mm membrane length, 100 kd cutoff, outer diameter 0.6 mm; CMA Microdialysis AB, Stockholm, Sweden) were introduced, one into each of the 4.0 endotracheal tubes, and guided by bronchoscopy into the distal bronchi of the left lung and right lung, respectively (Fig. 2) (9).

F2-13
Fig. 2:
Placement of the bronchial microdialysis catheters. The bronchoscope was advanced through the rubber diaphragm on the swivel adapter to the end of the endotracheal tube. One microdialysis catheter was advanced through one of the 4.0 endotracheal tubes until the shaft with the membranous tip was identified. The microdialysis catheter was then advanced by an assistant and guided by the bronchoscope into the selected main bronchus and further until it wedged. The position of the microdialysis catheters was controlled by bronchoscopy. Bronchoscopic examination controlled that the microdialysis catheter followed one of the major bronchi at each division point. If the microdialysis catheter was wedged in a side branch, the catheter was carefully retracted and guided into one of the major bronchi until the bronchoscope wedged and the microdialysis catheter disappeared in a small bronchus. The procedure was repeated to position the second microdialysis catheter. Finally, the bronchoscope was retracted to the trachea, and the microdialysis catheters were wedged and retracted approximately 0.5 cm.

One microdialysis catheter was inserted into the lumen of the jejunum 40 cm distal of the ligament of Treitz for cytokine measurements (20). In three of the pigs in the ischemia group, a microdialysis catheter for measuring fluorescein isothiocyanate Dextran 4,000 d (FD-4) was introduced into a jejunal loop 60 to 90 cm distal of the ligament of Treitz. The loop was closed at each end with ligatures and flushed with 50 mL Ringer acetate until the fluid was clear by visual control. Detection of FD-4 in the microdialysate was used as a marker of alterations in intestinal permeability.

Another two microdialysis catheters were inserted into the left and right subclavian arteries through venous catheters (Optiva2 18G, Medex, UK) (9).

One microdialysis catheter in each location (bronchial, arterial, and intestinal) was perfused at a flow rate of 1 μL/min with sterile phosphate buffered saline with 0.05% Tween for cytokine measurements. The other microdialysis catheters in the same locations were perfused with Plasmodex (Dextran 60, 30 g/1,000 mL; MEDA, Sweden) for FD-4 measurements. All microdialysis catheters were perfused in situ for 60 min or longer before the experiment was started. Samples from each microdialysis catheter were collected in 60-min fractions in microvials placed on ice 10 cm above the operating table. All microdialysis samples were analyzed immediately on-site for lactate and urea concentrations by enzymatic fluorometric assays (CMA600; CMA Microdialysis AB) using peroxidase methodology (9, 20). Cytokine samples were stored at −80°C. The FD-4 samples were stored at 4°C wrapped up in aluminum foil to avoid exposure to light.

Cytokine analysis

Microdialysis and arterial plasma samples were analyzed for TNF-α, IL-1β, and IL-8 by pig-specific bead-based assay on a Luminex 100 with the XY platform as previously reported (21). The method was modified as the time of reaction for the sample/microparticle mix was increased from 1 to 2 h. The limit of detection, mean fluorescence intensity ± 3 SD for blank samples, was 20 pg/mL for IL-1β, 20 pg/mL for IL-8, and 100 pg/mL for TNF-α.

Analysis of permeability by FD-4

The FD-4 is a macromolecule (polysaccharide) routinely used for measuring permeability and permeability changes in lung epithelium (22). The fluorescence of the plasma and microdialysate samples was measured on a fluorescence spectrophotometer (Fluoroskan II, Labsystems) (23). Samples were analyzed in duplicate with a volume of 25 μL. The method was modified using white opaque 96-well microplates to prevent background fluorescence from neighboring wells.

Lactate and urea measurements

All microdialysis samples were analyzed immediately on-site for lactate and urea concentrations by enzymatic fluorometric assays (CMA600, CMA Microdialysis AB) using peroxidase methodology.

Serum bioassay

The IL-6 activity in serum from the pulmonal arterial blood was determined with IL-6-dependent mouse hybridoma cell line B.13.29 clone 9 and calibrated with human IL-6 as described by Aarden et al. (24). All values are given as equivalents of the human standard curve.

Histology

At the end of the experiment, in all animals, a lung specimen was collected from the right diaphragmatic lobe through an anterior transdiaphragmatic access from the abdominal cavity.

Hematoxylin-eosin-stained sections were examined for alveolar hemorrhage, fibrin deposition, thickening of alveolar septa, vascular congestion, and neutrophil granulocytes in 10 random areas at 10× magnification in each section. Neutrophil granulocytes were counted, and the other findings were scored on a semiquantitative scale (0, no injury; 1, mild injury; 2, moderate injury; and 4, severe injury), modified from Douzinas et al. (15). The M30 Cytodeath (Roche, Basel, Switzerland) antibody recognizes a specific caspase cleavage site within cytokeratin 18 present in early apoptosis. Apoptotic cells were counted in 10 random areas at 10× magnification in each section. Areas with bronchial tissue were excluded in histological examinations.

Statistics and calculations

All values are presented as median and interquartile range when not otherwise mentioned. To assess changes within the groups over time, the Friedman test was used. Wilcoxon signed rank test was used to compare different time points within groups. Mann-Whitney U test was used to compare groups. Fisher exact test was performed on binomial data.

The individual arteriobronchial urea gradients for each period were used to correct the bronchial microdialysate concentrations of IL-1β, TNF-α, IL-8, and FD-4 for the area of microdialysis catheter membrane in contact with the epithelial lining fluid (9, 25).

(Ureaarterial/Ureabronchial)/(Moleculearterial/Moleculebronchial)

A microdialysate volume of 50 μL was used for bead-based multiplex cytokine measurements. In vials with volumes less than 50 μL, a dilution factor was calculated. SPSS for Mac 16.0.2 (Chicago, Ill) was used for the statistical analysis.

RESULTS

All animals in both groups survived the experimental period.

Hemodynamic and respiratory measurements

Hemodynamic and respiratory data are presented in Table 1.

T1-13
TABLE 1:
Systemic respiratory and circulatory variables in a model of 120 min of ischemia and 240 min of reperfusion of the superior mesenteric artery

The control animals remained stable as measured by heart rate, mean arterial pressure, and pulmonary capillary wedge pressure. Hemodynamic and respiratory values in both groups remained within the normal range for pigs, with a few exceptions (16). In the ischemia group during reperfusion, mean pulmonary artery pressure and pulmonary vascular resistance increased to values higher than reference values for conscious pigs. Heart rate, mean arterial pressure, and cardiac output are lower than reference values in conscious pigs mainly caused by the cardiodepressive effects of general anesthesia (26, 27).

Cytokines in microdialysis samples

In the ischemia group, there were individual differences in the concentrations of bronchial IL-8 and IL-1β during reperfusion, but the control group remained homogeneous (Fig. 3, A and B).

F3-13
Fig. 3:
Individual bronchial urea-corrected IL-8 and IL-1β. Individual values of bronchial urea-corrected IL-8 (A) and bronchial urea-corrected IL-1β (B) measured by microdialysis. The symbols are filled circles for the intestinal ischemia group and open circles for the sham-operated control group. On the abscissa baseline is the 60-min baseline period. The break on the abscissa was 120 min during which the control group was observed while the ischemia group received 120 min of ischemia of the superior mesenteric artery. Samples are plotted at the end of the sampling period.

The IL-8 and IL-1β in the ischemia group increased during the reperfusion phase as compared with baseline (P < 0.001 and P < 0.001, respectively), and there were significant increases between different time points and between the ischemia group and the control group (Fig. 4, A and B). Already within 1 h of reperfusion, intestinal ischemia induced increased bronchial epithelial lining fluid levels of both IL-8 and IL-1β (P = 0.018 and P = 0.012, respectively). Most bronchial TNF-α samples were below the limit of detection in both groups (data not shown). This may be caused by a high limit of detection for TNF-α in the present study.

F4-13
Fig. 4:
Bronchial urea-corrected IL-8 and IL-1β median ± interquartile range by microdialysis. Median values ± interquartile range of bronchial urea-corrected IL-8 (A) and bronchial urea-corrected IL-1β (B) measured by microdialysis. The symbols are filled circles for the intestinal ischemia group and open circles for the sham-operated control group. On the abscissa baseline is the 60-min baseline period. The break on the abscissa was 120 min during which the control group was observed while the ischemia group received 120 min of ischemia of the superior mesenteric artery. Values are plotted at the end of the sampling period. *P < 0.05 vs. the control group; † P < 0.05 vs.baseline; ‡ P < 0.05 vs. 60 min of reperfusion; § P < 0.05 vs. 120 min of reperfusion.

Luminal intestinal IL-8 increased in the ischemia group as compared with baseline (P = 0.003) and the control group (Fig. 5). The measured levels of intestinal IL-1β were low, and there were no changes during reperfusion compared with baseline. Intestinal TNF-α were below the limit of detection in all samples in both groups. This may be caused by a high limit of detection for TNF-α in the present study.

F5-13
Fig. 5:
Intestinal IL-8 median ± interquartile range by microdialysis. Median values ± interquartile range of luminal intestinal IL-8 measured by microdialysis. The symbols are filled circles for the intestinal ischemia group and open circles for the sham-operated control group. On the abscissa baseline is the 60-min baseline period. The break on the abscissa was 120 min during which the control group was observed while the ischemia group received 120 min of ischemia of the superior mesenteric artery. Values are plotted at the end of the sampling period. *P < 0.05 vs. the control group; † P < 0.05 vs. baseline.

Cytokine levels obtained by arterial microdialysis were mostly below the limit of detection and were not suitable for statistical analysis.

Cytokines in plasma samples

All samples in all animals were below the limit of detection.

Cytokines in pulmonary arterial serum

During reperfusion, the concentration of IL-6 in serum increased in the ischemia group, 130 (33-315) pg/mL as compared with the control group, 4 (0-30) pg/mL (P = 0.001).

The concentration of IL-6 was elevated (>10 pg/mL) in two of the animals at baseline and at some of the reperfusion time points in the control group. This was most likely caused by the trauma of the sham operation (tracheostomy, laparotomy, and preparation of the superior mesenteric artery) and positive-pressure ventilation (28).

Lactate levels in microdialysis samples

Luminal intestinal lactate by microdialysis increased during reperfusion in the ischemia group from baseline, 0.40 (0.15-0.52), to reperfusion, 3.97 (3.08-4.78) (P = 0.012). Bronchial lactate by microdialysis was the same in both groups.

Permeability by FD-4

Intestinal luminal FD-4 was increased in the ischemia group during reperfusion, 3.16 (1.58-4.82) μg/mL, as compared with the control group, 0.91 (0.56-2.06) (P = 0.002). This is in accordance with previous findings from our group and is an indication of the intestinal barrier dysfunction after intestinal ischemia-reperfusion injury (23).

Bronchial FD-4 did not increase in response to intestinal ischemia. However, we observed a difference in FD-4 baseline levels between the bronchi, intestinal lumen, and arterial blood as measured by microdialysis. Baseline microdialysis of FD-4 concentrations differed between bronchi and intestinal lumen. The baseline FD-4 concentration in the bronchi was approximately one half of the arterial concentration; and in the intestinal lumen, the concentration was less than one tenth of the arterial concentration (P < 0.001).

Histology and immunohistochemistry

There was no sign of diffuse alveolar damage by analysis of hematoxylin-eosin-stained sections. Neither the control group nor the ischemia group revealed any sign of edema, congestion, bleeding, and leakage into the alveoli or increased neutrophil count.

No significant change in the number of apoptotic cells as indicated by immunohistochemistry, Cytodeath M30, was detected.

Fluid recovery by microdialysis

There were no missing samples in the microdialysis catheters perfused with crystalloids. The fluid recovery in 95% of the samples from catheters perfused with crystalloids was greater than 65%.

The colloid perfusion fluid used in the microdialysis for FD-4 improved fluid recovery as compared with crystalloid perfusion fluid used for collection of cytokines in bronchi (P < 0.001) and in intestines (P < 0.001) (Table 2).

T2-13
TABLE 2:
Fluid recovery by microdialysis

DISCUSSION

The major finding in this study was the immediate detection and quantification of IL-1β and IL-8 in the bronchial epithelial fluid by microdialysis during reperfusion after 120 min of intestinal ischemia. In addition, bronchial microdialysis measured individual patterns of increasing levels of the proinflammatory cytokines IL-1β and IL-8 in the ischemia group throughout the early reperfusion period. Neither conventional histological nor immunohistochemistry techniques were able to identify lung tissue injury or apoptosis in the present experimental model at this early stage. The onset of early secondary lung involvement during intestinal reperfusion was implied by modest changes in a few hemodynamic and respiratory parameters, most of them within reference values (16). Only mean pulmonary artery pressure and pulmonary vascular resistance in the ischemia group increased higher than reference values during reperfusion. The shunt increased in the ischemia group during reperfusion as compared with the control group. There was also a slight reduction in PaO2/FiO2 ratio in the reperfusion period in the ischemia group. Our data showed that even before onset of manifest lung injury in intestinal ischemia and reperfusion, bronchial microdialysis identified individual patterns of increased cytokine levels.

Previous studies of airway cytokines, mainly by bronchoalveolar lavage techniques, suggest that IL-1β is a major proinflammatory cytokine present in the early phase of ARDS, and that IL-1β is also partially responsible for the increased permeability across lung epithelial and endothelial layers via interference with integrins (4, 29). Some studies indicate that IL-1β may even be important in the repair process of alveolar epithelium (30). The IL-8 is identified as the major chemokine that attracts and activates neutrophils. In alveoli and bronchi, the epithelial cells and alveolar macrophages produce IL-8 at an early stage of ALI/ARDS (4, 31). The expression of IL-8 mRNA has been found to appear as early as 2 min after lung reperfusion (32). In accordance to this finding, we were able to detect increased levels of bronchial IL-8 and IL-1β even during the first 60 min of reperfusion after a secondary lung insult by intestinal ischemia-reperfusion, with a further increase throughout the remaining reperfusion period.

Although there was a significant increase in bronchial IL-8 and IL-1β in the ischemia group, the individual response varied widely between subjects. Patients exposed to major trauma recover, experience serious comorbidity with sequela, or die; and individual levels of cytokines at different time points have been correlated to severity of ALI/ARDS and outcome (33). Our data identify individual profiles of the immediate bronchial cytokine response to intestinal ischemia-reperfusion injury. The animals in the control group showed homogeneity and remained stable. In the ischemia group, we found heterogeneous patterns of IL-1β and IL-8; some individuals with high cytokine levels and linear increase, increasing and then decreasing levels of cytokines, late increase and individuals with the same concentrations of bronchial cytokines as the control group. Our study does not answer the question of when to measure and for how long. But bronchial microdialysis allows us to continuously identify and quantify the individual cytokine response that may be correlated to individual morbidity and mortality. The present results suggest bronchial microdialysis as a possible tool for correlating individual cytokine profile to illness severity and outcome.

The arterial concentrations of TNF-α, IL-1β, and IL-8 measured by microdialysis were generally low and did not differ between the ischemia and the sham-operated control group. Plasma samples of TNF-α, IL-1β, and IL-8 were all below the limit of detection. This is in accordance with present research showing low levels of IL-1β, TNF-α, and IL-8 in blood, with no significant increase compared with controls in nonseptic multiple organ failure (34). In our experimental intestinal ischemia-reperfusion model of secondary lung inflammatory response, the major cytokine response occurred within the bronchi and, therefore, should be monitored within the bronchi and not in the blood.

The cytokine response in the primary injured organ, the intestine, differed from that seen in the lungs. Instead of a gradual increase in cytokine concentration in the bronchial epithelial lining fluid, the level of intestinal IL-8 was at maximum in the first reperfusion period, with no further increase during the rest of the reperfusion period. The inflammatory response evolves during the 120 min of ischemia and is manifest at the onset of reperfusion, with no further attenuation during late reperfusion (17). There rather seems to be a repair process and regeneration of injured epithelium during reperfusion (35). The systemic and secondary organ response to an ischemic trauma is small during ischemia but manifests and increases significantly during early reperfusion (36). Microdialysis allows a detailed profile of the inflammatory responses of both the primary organ (intestine) and the secondary organ (lung) for defined periods.

Baseline intestinal IL-8 values were about 2 ng/mL. This may reflect manipulation of the gut, but this is not known at present. One study on the supernatant from duodenal cell cultures obtained from a control group in a study on humans gave IL-8 values between 0 and 10 ng/mL (37). Interleukin-1β values were low in baseline, with no further increases. Nanogram concentrations of IL-8 in the lumen of the small intestine can be normal but need to be confirmed by further studies.

Serum bioassay of IL-6 showed an increase during reperfusion after ischemia (in accordance with findings in previously performed pilot studies by our group; data not shown). Interleukin-6 is an early acute-phase cytokine distributed by endocrine (liver) or paracrine (endothelium) mechanisms. Higher concentrations of IL-6 in blood are correlated to increased morbidity and mortality in clinical and experimental studies on reperfusion injury (33, 34). The increased IL-6 bioactivity in serum despite the undetectable/low plasma concentrations of TNF-α, IL-1β, and IL-8 indicates that our model of intestinal ischemia and reperfusion generates a systemic inflammatory response with possible involvement of distant organs.

The FD-4 has been evaluated as a marker molecule of paracellular leakage in an isolated lung model (38). In the present study, there was no difference in plasma-to-bronchial ratio of FD-4 between the ischemia group and the control group. The unchanged blood-bronchial ratio of FD-4 in the ischemia group indicated that there was no change in leakage and transport through the paracellular blood-bronchial barrier in the early reperfusion period after intestinal ischemia.

Intestinal ischemia induces an increased intestinal permeability during intestinal reperfusion, and increased luminal intestinal lactate is correlated to increased intestinal barrier dysfunction (39). The plasma-to-lumen ratio of FD-4 calculated from arterial and luminal intestinal microdialysis samples and increased levels of luminal lactate verified the primary intestinal barrier dysfunction as previously described by our group (23). The intestinal data confirm that the model induces intestinal barrier dysfunction as intended.

Microdialysis to allow the collection of cytokines with semipermeable membranes with larger pores introduces the challenge of fluid recovery. In vitro studies have revealed the benefit of positioning the collecting vials below the microdialysis catheter membrane (gravity) and adding colloids as albumin or Dextran to improve fluid recovery, but the effect in vivo is not verified (11, 40). The use of Dextran 60 or albumin 3.5% has shown a fluid recovery of about 100% in microdialysis in the central nervous system (11). In our study, a crystalloid (phosphate buffered saline with 0.05% Tween) was used as perfusion fluid for collecting cytokines and a colloid (30 g/L Dextran 60, Plasmodex) perfusion fluid was used to collect FD-4. We had no empty vials in either of the groups in arterial or bronchial or luminal intestinal microdialysis. Our study was not designed to measure the exact fluid recovery, but 50 μL was the standard volume for further analysis; and in the crystalloid samples we measured, the maximal recovered amount of fluid of the collected volume was less than 50 μL. There was a significant improved fluid recovery in the colloid group in luminal intestinal and bronchial microdialysis samples. In arterial microdialysis with a crystalloid perfusion, fluid recovery was less than 50 μL in only three vials. The reduced fluid recovery in these vials was caused by catheter membrane leakage caused by membrane injury during the insertion of the catheter. Further research is required to find the optimal perfusion fluid in bronchial and luminal intestinal microdialyses.

The relative recovery of a molecule by microdialysis allows us to estimate the concentration of this molecule at the site of the microdialysis membrane (12). The in vitro relative recovery percentages using crystalloid perfusion fluid of cytokines measured in our study were found to be about 35% for IL-1β, about 48% for IL-8, and about 4% for TNF-α (12). The low relative recovery of TNF-α (17 kd) is caused by the active protein being a trimer with a molecular weight of 52 kd, and the TNF-α data in the present study must be evaluated with this limitation. There is increasing evidence that the addition of human albumin solution of 0.1% to 3.5% into the perfusion fluid improves the in vitro relative recovery of IL-8 to about 73%, TNF-α to about 31%, leaving the relative recovery of IL-1β unchanged (12). The addition of albumin to improve relative recovery of cytokines in vivo remains to be evaluated.

The limited availability of reagents for flow cytometry in pigs (IL-1β, IL-8, and TNF-α) and the high limit of detection (IL-1β, 20 pg/mL; IL-8, 20 pg/mL; and TNF-α, 100 pg/mL) were concerns when the study was designed (21). However, human multiplex cytokine assays for flow cytometry contain a much larger number of proinflammatory and anti-inflammatory cytokines, allowing a detailed profiling of the inflammatory process, and the detection limit for cytokines is less than 10 pg/mL, and in some ultrasensitive kits, even less than 1.5 pg/mL. As an early study of microdialysis for sampling and analyzing cytokines from the bronchial epithelial fluid, we still feel confident that the three available cytokine reagents were sufficient to evaluate the usefulness of this technique, and a continuous cytokine profile for each pig was produced.

Microdialysis and multiplex cytokine assays open new research areas. The size and the speed of time-to-time changes and ratios between proinflammatory and anti-inflammatory cytokines are targets for further research.

CONCLUSIONS

This study on bronchial microdialysis of multiple cytokines successfully identified and quantified IL-1β and IL-8, but not TNF-α, during the first 4 h of reperfusion after intestinal ischemia. We found high concentrations of cytokines in the bronchial lumen and in the lumen of the small intestine in our pig model of intestinal ischemia and reperfusion. Arterial microdialysis and plasma IL-1β, IL-8, and TNF-α concentrations were generally low, with no group differences. The immediate inflammatory response seems to take place within the respective organs.

The positive cytokine findings in this study may be the first step toward an early and continuous monitoring of numerous important markers of inflammation in patients at risk for multiple organ failure to early determine the prognosis and the need for individual interventions before onset of manifest multiple organ failure. The potential clinical applicability of this method must be evaluated further.

ACKNOWLEDGMENTS

The authors thank senior engineer Unn Granli, Department of Laboratory Medicine, Children's and Women's Health, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), for the development of histological sections and application of the immunohistochemical method. Consultant Trond Viseth, Department of Pathology, St Olavs Hospital, conducted the histological analysis. Professor Eirik Skogvoll, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, was a discussion partner for the choice of statistical methodology in this study. Professor Terje Espevik and Engineer Liv Ryan, Department of Cancer Research and Molecular Medicine, Faculty of Medicine, NTNU, conducted the IL-6 bioassay analysis.

REFERENCES

1. McClintock D, Zhuo H, Wickersham N, Matthay M, Ware L: Biomarkers of inflammation, coagulation and fibrinolysis predict mortality in acute lung injury. Crit Care 12:R41, 2008.
2. Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F 2nd, Park DR, Pugin J, Skerrett SJ, Hudson LD, Martin TR: Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 164:1896-1903, 2001.
3. Parsons PE, Eisner MD, Thompson BT, Matthay MA, Ancukiewicz M, Bernard GR, Wheeler AP: Lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury. Crit Care Med 33:1-6, 2005 [discussion 230-232].
4. Pugin J, Verghese G, Widmer MC, Matthay MA: The alveolar space is the site of intense inflammatory and profibrotic reactions in the early phase of acute respiratory distress syndrome. Crit Care Med 27:304-312, 1999.
5. Huang YC, Bassett MA, Levin D, Montilla T, Ghio AJ: Acute phase reaction in healthy volunteers after bronchoscopy with lavage. Chest 129:1565-1569, 2006.
6. Klein U, Karzai W, Zimmermann P, Hannemann U, Koschel U, Brunner JX, Remde H: Changes in pulmonary mechanics after fiberoptic bronchoalveolar lavage in mechanically ventilated patients. Intensive Care Med 24:1289-1293, 1998.
7. Krause A, Hohberg B, Heine F, John M, Burmester GR, Witt C: Cytokines derived from alveolar macrophages induce fever after bronchoscopy and bronchoalveolar lavage. Am J Respir Crit Care Med 155:1793-1797, 1997.
8. Kodama T, Kanazawa H, Tochino Y, Kyoh S, Asai K, Hirata K: A technological advance comparing epithelial lining fluid from different regions of the lung in smokers. Respir Med 103:35-40, 2009.
9. Tyvold SS, Solligard E, Lyng O, Steinshamn SL, Gunnes S, Aadahl P: Continuous monitoring of the bronchial epithelial lining fluid by microdialysis. Respir Res 8:78, 2007.
10. Hunt J: Exhaled breath condensate: an overview. Immunol Allergy Clin North Am 27:587-596, 2007.
11. Hillman J, Aneman O, Anderson C, Sjogren F, Saberg C, Mellergard P: A microdialysis technique for routine measurement of macromolecules in the injured human brain. Neurosurgery 56:1264-1268, 2005 [discussion 1268-1270].
12. Helmy A, Carpenter KL, Skepper JN, Kirkpatrick PJ, Pickard JD, Hutchinson PJ: Microdialysis of cytokines: methodological considerations, scanning electron microscopy, and determination of relative recovery. J Neurotrauma 26:549-561, 2009.
13. Eisenberg EJ, Conzentino P, Eickhoff WM, Cundy KC: Pharmacokinetic measurement of drugs in lung epithelial lining fluid by microdialysis: aminoglycoside antibiotics in rat bronchi. J Pharmacol Toxicol Methods 29:93-98, 1993.
14. Spanos C, Papaconstantinou P, Spanos P, Karamouzis M, Lekkas G, Papaconstantinou C: The effect of l-arginine and aprotinin on intestinal ischemia-reperfusion injury. J Gastrointest Surg 11:247-255, 2007.
15. Douzinas EE, Kollias S, Tiniakos D, Evangelou E, Papalois A, Rapidis AD, Tsoukalas GD, Patsouris E, Roussos C: Hypoxemic reperfusion after 120 mins of intestinal ischemia attenuates the histopathologic and inflammatory response. Crit Care Med 32:2279-2283, 2004.
16. Hannon JP, Bossone CA, Wade CE: Normal physiological values for conscious pigs used in biomedical research. Lab Anim Sci 40:293-298, 1990.
17. Solligard E, Juel IS, Bakkelund K, Jynge P, Tvedt KE, Johnsen H, Aadahl P, Gronbech JE: Gut luminal microdialysis of glycerol as a marker of intestinal ischemic injury and recovery. Crit Care Med 33:2278-2285, 2005.
18. Chatterjee K, Parmley WW, Ganz W, Forrester J, Walinsky P, Crexells C, Swan HJC: Hemodynamic and metabolic responses to vasodilator therapy in acute myocardial infarction. Circulation 48:1183-1193, 1973.
19. West JB: Respiratory Physiology: The Essentials. Philadelphia, PA: Lippincott Williams & Wilkins, 2000.
20. Solligard E, Juel IS, Bakkelund K, Johnsen H, Saether OD, Gronbech JE, Aadahl P: Gut barrier dysfunction as detected by intestinal luminal microdialysis. Intensive Care Med 30:1188-1194, 2004.
21. Johannisson A, Jonasson R, Dernfalk J, Jensen-Waern M: Simultaneous detection of porcine proinflammatory cytokines using multiplex flow cytometry by the xMAP technology. Cytometry A 69:391-395, 2006.
22. Matsukawa Y, Lee VHL, Crandall ED, Kim K-J: Size-dependent dextran transport across rat alveolar epithelial cell monolayers. J Pharm Sci 86:305-309, 1997.
23. Juel IS, Solligard E, Tvedt KE, Skogvoll E, Jynge P, Beisvag V, Erlandsen SE, Sandvik AK, Aadahl P, Gronbech JE: Post-ischaemic restituted intestinal mucosa is more resistant to further ischaemia than normal mucosa in the pig. Scand J Clin Lab Invest 68:106-116, 2008.
24. Aarden LA, Groot ERD, Schaap OL, Lansdorp PM: Production of hybridoma growth factor by human monocytes. Eur J Immunol 17:1411-1416, 1987.
25. Rennard SI, Basset G, Lecossier D, O'Donnell KM, Pinkston P, Martin PG, Crystal RG: Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol 60:532-538, 1986.
26. Moffitt EA, Sethna DH: The coronary circulation and myocardial oxygenation in coronary artery disease: effects of anesthesia. Anesth Analg 65:395-410, 1986.
27. Deng CY, Yu XY, Kuang SJ, Rao F, Yang M, Shan ZX, Qian WM, Zhou ZL, Lin QX, Wu SL, et al.: Electrophysiological effects of ketamine on human atrial myocytes at therapeutically relevant concentrations. Clin Exp Pharmacol Physiol 35:1465-1470, 2008.
28. Evans C, Galustian C, Kumar D, Hagger R, Melville DM, Bodman-Smith M, Jourdan I, Gudgeon AM, Dalgleish AG: Impact of surgery on immunologic function: comparison between minimally invasive techniques and conventional laparotomy for surgical resection of colorectal tumors. Am J Surg 197:238-245, 2009.
29. Ganter MT, Roux J, Miyazawa B, Howard M, Frank JA, Su G, Sheppard D, Violette SM, Weinreb PH, Horan GS, et al.: Interleukin-1beta causes acute lung injury via alphavbeta5 and alphavbeta6 integrin-dependent mechanisms. Circ Res 102:804-812, 2008.
30. Geiser T, Jarreau PH, Atabai K, Matthay MA: Interleukin-1beta augments in vitro alveolar epithelial repair. Am J Physiol Lung Cell Mol Physiol 279:L1184-L1190, 2000.
31. Kurdowska A, Noble JM, Steinberg KP, Ruzinski JT, Hudson LD, Martin TR: Anti-interleukin 8 autoantibody: interleukin 8 complexes in the acute respiratory distress syndrome. Relationship between the complexes and clinical disease activity. Am J Respir Crit Care Med 163:463-468, 2001.
32. Rao JN, Clark SC, Ali S, Kirby J, Flecknell PA, Dark JH: Improvements in lung compliance after pulmonary transplantation: correlation with interleukin 8 expression. Eur J Cardiothorac Surg 23:497-502, 2003.
33. Kellum JA, Kong L, Fink MP, Weissfeld LA, Yealy DM, Pinsky MR, Fine J, Krichevsky A, Delude RL, Angus DC: Understanding the inflammatory cytokine response in pneumonia and sepsis: results of the Genetic and Inflammatory Markers of Sepsis (GenIMS) Study. Arch Intern Med 167:1655-1663, 2007.
34. Sperry JL, Friese RS, Frankel HL, West MA, Cuschieri J, Moore EE, Harbrecht BG, Peitzman AB, Billiar TR, Maier RV, et al.: Male gender is associated with excessive IL-6 expression following severe injury. J Trauma 64:572-578, 2008 [discussion 578-579].
35. Juel IS, Solligard E, Lyng O, Stromholm T, Tvedt KE, Johnsen H, Jynge P, Saether OD, Aadahl P, Gronbech JE: Intestinal injury after thoracic aortic cross-clamping in the pig. J Surg Res 117:283-295, 2004.
36. Kostopanagiotou G, Avgerinos E, Costopanagiotou C, Arkadopoulos N, Andreadou I, Diamantopoulou K, Lekka M, Smyrniotis V, Nakos G: Acute lung injury in a rat model of intestinal ischemia-reperfusion: the potential time depended role of phospholipases A(2). J Surg Res 147:108-116, 2008.
37. Ando T, Watanabe O, Ishiguro K, Maeda O, Ishikawa D, Minami M, Hasegawa M, Kondo S, Goto Y, Ohmiya N, et al.: Relationships between Helicobacter pylori infection status, endoscopic, histopathological findings, and cytokine production in the duodenum of Crohn's disease patients. J Gastroenterol Hepatol 23:193-197, 2008.
38. Huang YC, Piantadosi CA: Alveolar barrier function assessed by hydrophobic and hydrophilic fluorescent solutes in rabbit lung. Respir Physiol Neurobiol 133:153-166, 2002.
39. Solligard E, Juel IS, Spigset O, Romundstad P, Gronbech JE, Aadahl P: Gut luminal lactate measured by microdialysis mirrors permeability of the intestinal mucosa after ischemia. Shock 29:245-251, 2008.
40. Hamrin K, Rosdahl H, Ungerstedt U, Henriksson J: Microdialysis in human skeletal muscle: effects of adding a colloid to the perfusate. J Appl Physiol 92:385-393, 2002.
Keywords:

Lung injury; cytokines; interleukin-8; interleukin-1β; tumor necrosis factor-α; microdialysis; small intestine; ischemia; reperfusion injury

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