1 Division of Anesthesiology and Critical Care Medicine, Department of Surgery, Tottori University Faculty of Medicine, 36-1 Nishi-cho, Yonago, Tottori, 683-8504, Japan. 2 Malaria Vaccine Development Unit, Laboratory of Parasitic Disease, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA. 3 Department of Anesthesia, University of Pennsylvania, Philadelphia, PA, USA
*Corresponding author. E-mail: ishibe@grape.med.tottori-u.ac.jp
Accepted for publication: December 5, 2003
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Abstract |
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Methods. Lungs isolated from female white Japanese rabbits were perfused and divided into a basal (BAS) group (n=7, baseline measurement alone), a control (CONT) group (n=9, ventilated without lung collapse for 120 min) and an atelectasis (ATEL) group (n=9, lung collapsed for 55 min followed by re-expansion and ventilation for 65 min). Pulmonary vascular resistance (PVR) and the coefficient of filtration (Kfc) were measured at baseline and 60 and 120 min. At the end of perfusion, bronchoalveolar lavage fluid/plasma protein ratio (B/P), wet/dry lung weight ratio (W/D) and mRNA expressions of tumour necrosis factor (TNF)-, interleukin (IL)-1ß and myeloperoxidase (MPO) were determined.
Results. TNF- and IL-1ß mRNA were significantly up-regulated in lungs of the ATEL group compared with BAS and CONT, though no significant differences were noted in PVR, Kfc, B/P and W/D within and between groups. MPO increased at 120 min in CONT and ATEL groups.
Conclusion. Pro-inflammatory cytokines were up-regulated upon re-expansion and ventilation after short-period lung collapse, though no changes were noted in pulmonary capillary permeability.
Br J Anaesth 2004; 92: 55863
Keywords: gene, expression; lung, pulmonary capillary permeability; lung, re-expansion; polypeptides, inflammatory cytokine
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Introduction |
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Previous animal studies from our laboratory7 demonstrated that 60-min lung collapse combined with perfusion arrest and 60-min follow-up after reperfusion and re-expansion-induced pulmonary oedema was accompanied by increased tumour necrosis factor (TNF)- concentrations in the perfusate in rats. However, it is impossible to distinguish a specific aetiology from these studies; i.e. whether perfusion arrest or lung collapse contributes to the induction of pulmonary oedema.
In the present study, we investigated the effects of short-term lung collapse (continuing pulmonary circulation) and re-expansion by measuring pulmonary vascular resistance, pulmonary capillary permeability and especially messenger RNA (mRNA) expression of pro-inflammatory cytokines by using isolated perfused rabbit lungs.
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Materials and methods |
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The isolated lungs were positioned on an electronic scale (model GX4000; A&D Co., Tokyo, Japan) placed in a box, to allow continuous monitoring of lung weight. After stabilization of perfusion and ventilation for 20 min, baseline pulmonary arterial pressure (Ppa), pulmonary venous pressure (Ppv) and coefficient of filtration (Kfc) were recorded. After baseline measurements, the lung preparations were divided at random into three groups. In the basal group (BAS; n=7), ventilation and perfusion were terminated without further intervention. In the control group (CONT; n=9), ventilation and perfusion were maintained for 120 min. In the atelectasis group (ATEL; n=9), the lungs were continuously perfused but completely collapsed for 55 min, followed by rapid re-inflation, and ventilation using the same conditions for 65 min. In the CONT and ATEL groups, the data were obtained at 60 and 120 min from baseline measurement. The perfusate samples were collected at 55 and 120 min. After termination of perfusion and ventilation, both lungs were used for further measurement.
Northern blot analysis
Experiments were performed in six rabbits in each of the three groups. Just after the end of perfusion, the lung tissue samples were collected from two locations in the right lung; the dorsal medial lobe and ventral superior lobe, and total RNA was obtained. Northern blot analysis was performed as described previously.10 11 In brief, total RNA 10 µg was separated on agarose-formaldehyde gel 1% and subsequently transferred and blotted onto INC nylon membranes (Westborough, MA, USA). Probes were prepared to purify products using the polymerase chain reaction with the primers listed in Table 1 for rabbit TNF-, interleukin (IL)-1ß, transforming growth factor (TGF)-ß1 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).1214 Each probe was labelled with [32P]dCTP and gene expression levels were analysed with a laser densitometer (Molecular imager; Bio-Rad GS-525; Japan Bio-Rad-laboratories, Tokyo, Japan). The TNF-
, IL-1ß and TGF-ß1 densitometer data were corrected for GAPDH and then expressed using the sum total of three groups as 100%. For each group, data obtained from the dorsal middle and ventral superior lobes were combined and analysed statistically.
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Rpa=(PpaPdo)Q1
Rpv=(PdoPpv)Q1
Rt=(Ppa Ppv)Q1
where Rt=total vascular resistance, Rpa=arterial vascular resistance, Rpv=venous vascular resistance and Pdo=double occlusion pressure.
Pulmonary capillary filtration coefficient
Kfc was determined according to the method of Drake et al.15 The venous reservoir was lifted by 6 cm H2O and the change in lung weight was measured continuously with an electronic balance. The change in lung weight per unit time (Wt
t1) was calculated using data from 120 to 360 s and the initial change was determined as [(
Wt
t1)t=0].
Pdo represented the difference in Pdo measured before and at 6 cm H2O pressure load. Kfc was calculated per 100 g of lung weight using the following formula:
Kfc=[(Wt
t1)t=0]
Pdo1 100 g1 (ml min1 mm Hg1 100 g1)
Wet to dry lung weight ratio (W/D)
The W/D of the right lung was measured as described previously.7 8 The ratio was calculated as W/D=wet weight/dry weight.
Collection of bronchoalveolar lavage fluid (BALF)
All rabbit lungs were used for analysis of BALF. Briefly, physiological saline solution (5 ml) at 4°C was injected into the left lung and BALF was subsequently collected. This procedure was repeated four times. The recovery rate of BALF was about 60%. Total protein concentrations in the BALF and perfusate were determined with BAC Protein Assay Reagent Kit (Pierce, Rockford, IL, USA) and BALF/plasma total protein ratio was calculated.
Myeloperoxidase (MPO) activity
MPO was measured in the perfusate and BALF at baseline and at 60 and 120 min using the method of o-dianisidine dihydrochloride oxidation.16
Statistical analysis
All parametric data are expressed as mean (SD). Effects of intervention on blood gas data, pulmonary vascular pressure and resistance, Kfc, airway pressure and MPO in the perfusate were analysed with Friedmans test and subsequent post hoc StudentNewmanKeuls (Stat View 5.0; SAS Institute, NC, USA). Levels of mRNA expression, BALF/plasma total protein ratio, W/D and MPO concentration in BALF were compared using the KruskalWallis H-test and subsequent post hoc StudentNewmanKeuls. A P value less than 0.05 denoted the presence of a statistically significant difference.
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Results |
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MPO activity
There were no differences in MPO activity in BALF between the three groups (BAS, 0.34 (0.24); CONT, 0.16 (0.04); ATEL, 0.27 (0.06) mol ml1 min1). MPO activity increased gradually but the change was similar in the CONT and ATEL groups (Table 4).
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Discussion |
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Our model has two physiological characteristics. The first is that pulmonary perfusion flow rate is kept constant during lung collapse. If collapsed and non-collapsed areas coexist in the same lung, blood flow should shift from the collapsed to the non-collapsed areas and the collapsed lung should become relatively ischaemic, because of a difference in blood vessel resistance.17 In our model, however, perfusion of the collapsed lungs was maintained, and thus the effect of ischaemia induced by hypoperfusion was excluded. Blood supply to the lung consists of two circulatory systems: the pulmonary and bronchial circulations. In our model, bronchial circulation was completely sacrificed. We considered that this effect on the present study results may have been negligible, as the pulmonary circulation is apparently dominant compared with the bronchial circulation. In addition, the pulmonary circulation flow rate was kept constant by using a pump. The second feature of our experimental design was the short period of alveolar collapse (55 min) and follow-up for 60 min of ventilation after re-expansion. These periods were used in order to compare the data with those of previous studies,7 8 in which pulmonary oedema was observed after 60 min collapse and perfusion arrest followed by 60 min of perfusion and ventilation. The 60-min follow-up time was also based on the finding that 64% of re-expansion pulmonary oedema occurs within 1 h after re-expansion.4 OLV with lung collapse for 1 or 2 h is frequently used in thoracic surgery, such as lobectomy, in the clinical setting. Fifty five-minute lung collapse followed by re-expansion in the present study did not change the physiological indexes of pulmonary oedema (Kfc, W/D, BALF/plasma total protein ratio and PVR). These results are different from those reported by Liu et al.7 8 They demonstrate that short-period lung collapse combined with perfusion arrest resulted in increases in physiological indices of lung oedema.7 8 The discrepancy in the results of the two studies indicates that the effect of short-period lung collapse while maintaining pulmonary circulation is less invasive than that occurring when combined with circulatory arrest, and does not lead to serious deterioration of lung function.
However, it is interesting that our results clearly showed up-regulation of mRNAs of the pro-inflammatory cytokines TNF- and IL-1ß only with 1-h collapse with perfusion and 1-h follow-up. In this study we focused on the expression of TNF-
and IL-1ß because these are typical early phase pro-inflammatory cytokines and are known to be up-regulated within the first 2 h. In addition, TNF-
and IL-1ß per se not only induce apoptosis and injure organs18 19 but also trigger cytokine networks and further activate additional cytokines, and attack cells.20 21 The present results suggest that TNF-
and IL-1ß may be involved in pulmonary oedema.13 Another inflammatory cytokine, IL-8, correlates with REPE,22 23 is up-regulated by TNF-
and IL-1ß 24 25 and shows sustained activity over several hours. As this study was designed to finish within 2 h, we did not measure IL-8. In contrast to TNF-
and IL-1ß, which were induced immediately after stimulation, TGF-ß1 was not induced in this study.26 We think that, because TGF-ß1 is a relatively late-phase cytokine,27 2 h is not enough time to induce TGF-ß1 in our study model.
MPO is an enzyme induced by activated neutrophils. Our results showed a 1.5-fold increase in MPO activity in the perfusate of the CONT and ATEL groups at the end of the study compared with baseline values. These findings suggest induction of neutrophil activation by surgical manipulation and subsequent perfusion rather than by lung collapse and re-expansion. On the other hand, there were no differences in BALF concentrations of MPO between the three groups, suggesting that neutrophils had not yet infiltrated into the alveolus.
In the clinical setting, OLV has become a common procedure for thoracoscopic surgery and in most cases it is performed without postoperative pulmonary complications. However, cases of REPE are reported after video-assisted thoracoscopic surgery.13 A possible mechanism is the vascular endothelial damage after reperfusion injury in the areas of previous hypoxic vasoconstriction, with the resulting reactive oxygen species altering the permeability of the vascular endothelium. Therefore, the duration and extent of exposure to hypoxia may play an important role in the development of REPE. In the present study, short-term lung collapse and re-expansion up-regulated the mRNAs of pro-inflammatory cytokines, which may increase cytokine activity and subsequent pulmonary capillary permeability, though lung oedema was not observed. As previously discussed, in the clinical setting blood flow to the collapsed lung decreases and the lungs become more ischaemic than in the present study. The duration of lung collapse in thoracoscopic surgery is usually longer than 60 min. Although there are limitations in extrapolation of the findings obtained from this ex vivo animal study, these considerations suggest that re-expansion of the lungs after OLV makes them susceptible to REPE.
In conclusion, we have demonstrated in the present study that short-period lung collapse and re-expansion up-regulated the pro-inflammatory cytokine expression despite the lack of physiological lung injury in perfused rabbit lungs.
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Acknowledgements |
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